Systems and methods for monitoring tissue sample processing

ABSTRACT

A tissue sample that has been removed from a subject can be evaluated. A change in speed of the energy traveling through the sample is evaluated to monitor changes in the biological sample during processing. The monitoring can detect movement of fluid with the sample and cross-linking. A system for performing the method can include a transmitter that outputs the energy and a receiver configured to detect the transmitted energy.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part under 35 U.S.C. § 120of U.S. application Ser. No. 13/372,040, filed on Feb. 13, 2012, whichis a continuation-in-part of International Application No.PCT/US2011/027284, filed on Mar. 4, 2011 (now converted to U.S.application Ser. No. 13/582,705, filed Sep. 4, 2012), which claimsbenefit under 35 U.S.C. § 119(e) of U.S. provisional application Ser.No. 61/310,653, filed on Mar. 4, 2010; and U.S. application Ser. No.13/372,040 also claims benefit under 35 U.S.C. § 119(e) of U.S.provisional application Ser. No. 61/464,479, filed on Mar. 4, 2011, andU.S. provisional application Ser. No. 61/463,551, filed on Feb. 17,2011. All of these prior applications are incorporated herein byreference in their entireties.

TECHNICAL FIELD

The present disclosure relates generally to systems and methods foranalyzing specimens. More specifically, the present disclosure relatesto methods and systems for monitoring processing of tissue samples.

BACKGROUND

Preservation of tissues from surgical procedures is currently a topic ofgreat importance. Currently, there are no standard procedures for fixingtissues and this lack of organization leads to a variety of stainingissues both with primary and advanced stains. The first step afterremoval of a tissue sample from a subject is to place the sample in aliquid that will suspend the metabolic activities of the cells. Thisprocess is commonly referred to as “fixation” and can be accomplished byseveral different types of liquids. The most common fixative in use byanatomical pathology labs is 10% neutral buffered formalin (NBF). Thisfixative forms cross-links between formaldehyde molecules and aminecontaining cellular molecules. In addition, this type of fixativepreserves proteins for storage.

When used at room temperature, NBF diffuses into a tissue section andcross-links proteins and nucleic acids, thereby halting metabolism,preserving biomolecules, and readying the tissue for paraffin waxinfiltration. The formalin can be at slightly elevated temperature(i.e., higher than room temperature) to further increase thecross-linking rate, whereas lower temperature formalin can significantlydecrease the cross-linking rate. For this reason, histologists typicallyperform tissue fixation at room temperature or higher. Some groups haveused cold formalin, but only in specialized situations and not forfixing tissues. For instance, cold formalin has been used to examinelipid droplets.

Several effects are often observed in tissues that are either underexposed or over exposed to formalin. If formalin has not diffusedproperly through the tissue samples, outer regions of the tissue samplesexposed to formalin may be over-fixed and interior regions of the tissuesamples not exposed to formalin may be under-fixed, resulting in verypoor tissue morphology. In under-fixed tissue, subsequent exposure toethanol often shrinks the cellular structures and condenses nuclei sincethe tissues will not have the chance to form a proper cross-linkedlattice. When under-fixed tissue is stained, such as with hematoxylinand eosin (H&E), many white spaces may be observed between the cells andtissue structures, nuclei may be condensed, and samples may appear pinkand unbalanced with the hematoxylin stain. Tissues that have beenexposed to excess amounts of formalin or too long typically do not workwell for subsequent immunohistochemical processes, presumably because ofnucleic acid and/or protein denaturation and degradation. As a result,the optimal antigen retrieval conditions for these tissues do not workproperly and therefore the tissue samples appear to be under stained.

Proper medical diagnosis and patient safety often require properlyfixing the tissue samples prior to staining. Accordingly, guidelineshave been established by oncologists and pathologists for properfixation of tissue samples. For example, according to the AmericanSociety of Clinical Oncology (ASCO), the current guideline for fixationtime in neutral buffered formalin solution for HER2 immunohistochemistryanalysis is at least 6 hours, preferably more, and up to 72 hours. Itmay be advantageous to develop a process for monitoring diffusion offixatives through a tissue sample to determine whether the fixative hasinfused the entire tissue sample to minimize or limit under-fixed tissueor over-fixed tissue and to better preserve biological molecules, tissuemorphology, and/or post-translational modification signals beforesignificant degradation occurs.

Overveiw of Disclosure

At least some embodiments disclosed herein are methods of preparingspecimens for a fixation process. Specimens, such as solid tissuesamples, can be contacted with a liquid fixative that travels throughthe tissue samples. The fixative can be allowed to diffuse throughoutsubstantially the entire thickness of the tissue samples. After asufficient amount of fixative has infused the tissue samples, a fixationprocess can be performed to fix substantially all of the tissue, therebyminimizing or limiting under-fixation and over-fixation in, for example,outer regions and inner regions. The method can enhance the quality inpreservation of tissue methodology, protein structure, and/orpost-translation modification signals.

At least some embodiments disclosed herein are methods and systems foranalyzing a tissue sample based on its characteristics, includingacoustic characteristics, mechanical characteristics, opticalcharacteristics, or other characteristics that may be static or dynamicduring processing. In some embodiments, acoustic properties of thetissue sample are continuously or periodically monitored to evaluate thestate and condition of the tissue sample throughout processing. Based onthe obtained information, processing can be controlled to enhanceprocessing consistency, reduce processing times, improve processingquality, or the like.

Acoustics can be used to non-invasively analyze tissue samples. When anacoustical signal interacts with tissue, transmitted signals (e.g.,signals transmitted through the tissue sample) depends on severalmechanical properties of the sample, such as elasticity and firmness.The acoustic properties of tissue samples may change as liquid reagent(e.g., a liquid fixative) travels through the sample. In someprocedures, acoustic properties of the tissue sample can change asinterstitial fluid is displaced with liquid reagent because of differentacoustic properties between the interstitial fluid and liquid reagent.Even though fixatives may not result in substantial cross-linking, theacoustic properties of the tissue sample can change as the fixativetravels across the thickness of the sample. The sample's acousticproperties can change during, for example, a pre-soak process (e.g.,diffusion of cold fixative), a fixation process, a staining process, orthe like. In the fixation process (e.g., a cross-linking process), thespeed of transmission of acoustic energy can change as the tissue samplebecomes more heavily cross-linked.

In some embodiments, a method for tissue preparation can includecontacting a tissue sample with a fixative. Real-time monitoring can beused to accurately track movement of the fixative through the sample.After the fixative has diffused through the tissue sample, a fixationprocess and a subsequent histological process can be performed. A statusof a biological sample can be monitored based on a time of flight ofacoustic waves. The status can be a diffusion status, a density status,a fixation status, a staining status, or the like. Monitoring caninclude, without limitation, measuring changes in a level of diffusion,sample density, cross-linking, decalcification, stain coloration, or thelike. The biological sample can be solid or non-fluidic tissue, such asbone, or other type of tissue.

In some embodiments, methods and systems are directed to using acousticenergy to monitor a tissue sample. Based on interaction between theacoustic energy in reflected and/or transmission modes, informationabout the specimen may be obtained. Examples of measurements includeacoustic signal amplitude, attenuation, scatter, absorption, time offlight (TOF) in the specimen, phase shifts of acoustic waves, orcombinations thereof. In some procedures, a fixative is applied to thespecimen. As the fixative diffuses through the specimen, the tissuesample's mechanical properties (e.g., elasticity, stiffness, etc.)change, and these changes can be monitored using sound speedmeasurements via TOF. Subsequent processes can be monitored based on TOFand a state of the specimen (e.g., a state of saturation, a fixativestate, a histological state, etc.) can be determined. To avoidunder-fixation or over-fixation, the static characteristics of thetissue (including reagent(s) in the tissue), dynamic characteristics ofthe tissue, or both can be monitored. Characteristics of the tissueinclude transmission characteristics, reflectance characteristics,absorption characteristics, attenuation characteristics, or the like.

In some procedures, an unfixed tissue sample is contacted with afixative. The movement of the fixative through the tissue sample can bemonitored in real-time. The composition of the fixative can be selectedto enhance monitoring. For example, NBF has a relatively high bulkmodulus compared to interstitial fluid. The sound transmissibility ofthe fixative is related to its bulk modulus (k) and density (p)according to the speed equation,

${{speed}\mspace{14mu}{of}\mspace{14mu}{sound}\mspace{14mu}{in}\mspace{14mu}{fixative}} = {\sqrt{\frac{k}{p}}.}$The fixative, such as formalin, with a bulk modulus greater thaninterstitial fluid can significantly alter the TOF as it displaces theinterstitial fluid. Once a desired level of diffusion is achieved, thetissue sample can be removed from the fixative to generally stop furtherdiffusion. The tissue sample, including the fixative within the tissuesample, can be heated to promote cross-linking and fixation.

A TOF acquisition scheme can be used to monitor tissue samples. The TOFacquisition scheme can include an ND conversion scheme (e.g., about 1μsec phase comparison) to obtain a large number of phase comparisons toprovide generally real-time monitoring. The phase comparisons can beperformed at the same frequency and phase relationship, and thetemperature of the fixative and/or tissue sample can remain generallyconstant to increase signal to noise ratios. Because fluctuations intemperature may cause measureable changes in TOF, the TOF acquisitionscheme can compensate for changes in TOF attributable to, for example,temperature changes.

In some embodiments, a method for evaluating tissue sample includescontacting tissue sample with a reagent. Diffusion of the reagentthrough the tissue sample can be monitored based on properties of thetissue sample, including mechanical properties, acoustic properties,and/or optical properties. Monitoring includes, without limitation,measuring time of flight of acoustic waves that travel through a tissuesample. This monitoring can include, transmitting acoustic waves acrossa thickness of the sample while a reagent gradually moves across thetissue sample. After the reagent in the form of fixative has diffusedthrough most of the volume of the tissue sample, a fixation process isperformed. For example, the fixative can diffuse through at least 90% byvolume of the tissue sample. In other embodiments, the fixative candiffuse through substantially all the volume of the tissue sample. Inone procedure, the fixative can diffuse through at least 95% by volumeof the tissue sample. Such processes can substantially eliminateover-fixation or under-fixation of interior and outer regions of tissuesamples.

In some embodiments, a processing method comprises contacting a tissuesample, which is in an unfixed state, with a liquid fixative. Themovement of the liquid fixative through a tissue sample is acousticallymonitored. A fixation process can be performed after the liquid fixativehas displaced a target volume of interstitial fluid from the tissuesample. In one procedure, the fixation process is performed after theliquid fixative has displaced at least 50% by volume of the interstitialfluid. In other procedures, the fixation process is performed after thefixative has displaced at least 75% by volume of the interstitial fluid.Such fixation processes can include heating the tissue sample to promotecross-linking.

A processing system, in some embodiments, comprises an acousticmonitoring device and a computing device communicatively coupled to theacoustic monitoring device. The acoustic monitoring device can detectacoustic waves that have traveled through a tissue sample. The computingdevice can be configured to evaluate the speed of an acoustic wavetraveling through the tissue sample based on time of flight. Thecomputing device, in some embodiments, includes instructions formonitoring diffusion of a liquid using the acoustic monitoring deviceand for performing a fixation process. The acoustic monitoring device,in some embodiments, includes one or more transmitters and one or morereceivers. The tissue sample can be immersed in a liquid fixative whilethe transmitters and receivers communicate to detect time of flight ofacoustic waves.

In some embodiments, a method of evaluating a tissue sample comprisescontacting the tissue sample with a fixative. The movement of thefixative through the tissue sample can be monitored based on theacoustic waves. In certain procedures, the monitoring can includesimultaneously monitoring diffusion and cross-linking performed at thesame or different processing temperatures. For example, diffusion andcross-linking can be performed while a fixative and/or a tissue sampleis maintained at the same general temperature. In other procedures, thediffusion and cross-linking can be performed at different temperatures.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting and non-exhaustive embodiments are described with referenceto the following drawings. The same reference numerals refer to likeparts or acts throughout the various views, unless otherwise specified.

FIG. 1 is an isometric, cutaway view of a processing system containing aspecimen holder with a specimen, in accordance with one embodiment.

FIG. 2 is a side cross-sectional view of components of the processingsystem of FIG. 1.

FIG. 3 is a block diagram of components of an analyzer and a computingdevice, in accordance with one embodiment.

FIG. 4 is a flow diagram of an exemplary method of processing aspecimen, in accordance with one embodiment.

FIG. 5 is a graph of fixation phase versus change in time of flight.

FIG. 6 is a plot showing a timing relationship between an outputtedsignal and a received signal.

FIG. 7 is an enlarged view of a portion of the outputted signal and aportion of the received signal.

FIG. 8 is a detailed view of a portion of the outputted signal and acorresponding portion of the received signal.

FIG. 9 is a plot showing a timing relationship between an outputtedsignal, a received signal, and a comparison curve.

FIG. 10A is a plot showing a timing relationship between an outputtedsignal and a received signal, in accordance with one embodiment.

FIG. 10B is a plot showing a timing relationship between an outputtedsignal and a received signal, in accordance with yet another embodiment.

FIG. 11 is a graph of phase difference versus phase angle voltage and aplot showing time versus phase angle voltage with an expected phaseequal progression due to fixation.

FIG. 11A is a plot of frequency versus phase comparison in accordancewith one embodiment.

FIG. 12 is a plot of fixation time versus phase angle voltage.

FIG. 13 is a plot of fixation time versus phase angle voltage.

FIG. 14 is a plot showing jaggedness of the data of FIG. 13.

FIG. 15 is a plot of jaggedness generated using a smoothing algorithmand the data of FIG. 13.

FIG. 16 is a plot of curves generated using different algorithms foranalyzing noisy data.

FIG. 17 is a block diagram of a processing system, in accordance withone embodiment.

FIG. 18 is an isometric view of a processing system capable ofsequentially analyzing specimens.

FIG. 19 is an elevated, partial cross-sectional view of a processingsystem capable of performing multiple treatments on specimens, inaccordance with one embodiment.

FIG. 20 is a side elevational view of a processing system capable ofperforming multiple treatments on specimens, in accordance with oneembodiment.

FIG. 21 is a side elevational view of a processing system capable ofindividually processing tissue samples.

FIG. 22 is an isometric view of an analyzer with a rotary drive system.

FIG. 23 is a processing system capable of fixing and embedding a tissuesample.

FIG. 24 is a flow diagram of an exemplary method of processing aspecimen.

FIG. 25 is an isometric view of an analyzer ready to receive a specimenholder.

FIG. 26 is an isometric view of an analyzer with a linear array oftransmitters and a linear array of receivers.

FIG. 27 is an isometric view of a specimen holder, in accordance withone embodiment.

FIG. 28 is an isometric view of a specimen holder with transmitters andreceivers.

FIG. 29 is a side elevational view of the specimen holder of FIG. 28.

FIG. 30 is a plot of fixation time versus sound of speed in tissue andabsolute TOF change for beef tissue.

FIG. 31 is a plot of fixation time versus sound speed and relative TOFchange for beef tissue.

FIG. 32 is a plot of fixation time versus signal amplitude and TOFchange for beef tissue.

FIG. 33 is a plot of fixation time versus TOF change and signalamplitude for fat tissue.

FIG. 34 is a plot of fixation time versus signal amplitude and TOFchange for liver tissue.

FIG. 35 is a plot of fixation time versus signal amplitude and TOFchange of human tonsil tissue.

FIG. 36 is a plot of fixation time versus signal amplitude and TOFchanges for beef tissue.

FIG. 37 is a plot of fixation time versus signal amplitude for differenttypes of tissue.

FIG. 38 is a plot of fixation time versus change of TOF for differenttypes of tissues.

FIG. 39 is a plot of time versus a time of flight signal for a presoakedsample and a fresh sample.

FIG. 40 is a plot of time versus a time of flight signal for a fixationand dehydration process.

FIG. 41 is a plot of time versus amplitude of a time of flight signalfor insufficiently fixed tissue and fixed tissue.

FIG. 42 is a plot of time versus time of flight signal amplitude for atissue sample submerged for different lengths of time in formalin.

FIG. 43 is a plot of time of flight for water, 10% NBF, and 20% NBF.

FIG. 44 is a plot of time versus time of flight for tissue samplessubmerged in water, 10% NBF, and 20% NBF.

FIG. 45 is a plot of time versus time of flight for tissue samplesduring cold diffusion.

DETAILED DESCRIPTION

At least some embodiments of the present disclosure are directed tomonitoring a multi-step tissue preparation process that includes,without limitation, a fixative delivery process (“delivery process”) anda fixation/cross-linking process (“fixation process”). The deliveryprocess can include contacting the tissue sample with a liquid fixativeat a first temperature for a first period of time. Movement of thefixative can be monitored to evaluate whether the fixative hasadequately infused the sample. After desired diffusion is achieved, thetissue sample can be heated to a second temperature higher than thefirst temperature to start or promote cross-linking. If the fixative isformaldehyde, cross-linking can occur between formaldehyde molecules andamine containing cellular molecules without significantly compromisingthe tissue characteristics (e.g., antigenicity and/or morphology).

The delivery process can involve diffusion of cold fixative throughoutsubstantially the entire thickness or cross section of the tissuesample. The cold fixative can be at a temperature in a range of about−20° C. to about 15° C., preferably greater than 0° C. to an uppertemperature more typically about 10° C., and even more preferably fromabout 3° C. to about 5° C. For some procedures, the fixative temperaturecan be about 4° C. However, the cold fixative can be at othertemperatures. The delivery time period can be in a range of about 15minutes up to about 4 hours, most typically from greater than 15 minutesto about 3 hours, with desirable results typically being obtained byimmersing tissue samples for about 1.5 hours to about 2 hours.Increasing the delivery time period to 4 hours or greater may havelittle beneficial effect for smaller tissue pieces with thicknesses upto about, for example, 4 mm. Additionally, the composition of thefixative can be selected to achieve a desired diffusion coefficient.Example fixatives include aldehyde fixatives, such as 10% NBF or 20%NBF. Table 1 below shows diffusion rate constants for cores of tonsiltissue with about 6 mm thicknesses. The diffusion rate at a temperatureof 4° C. through 6 mm cores of tonsil tissue can be approximated bydiffusion equations or diffusion modeling.

TABLE 1 Rate Constant C NBF (at 4 C./6 mm Time to reach 50% to reach 95%Concentrations core) [hours] [hours] 10% 0.00015 1.3 5.5 20% 0.0002 1.04.2 40% 0.00025 0.8 3.3Table 1 shows that 40% NBF can diffuse through about 50% of the volumeof the 6 mm core tissue specimen in about 0.8 hour and about 95% of thetissue sample in about 3.3 hours. The 20% NBF can diffuse through about50% of the volume of the tissue specimen in about 1.3 hours and about95% of the tissue sample in about 5.5 hours. Accordingly, to reduceprocessing times, the concentration of NBF can be increased to increasethe rate of diffusion

Although some cross-linking may occur during the delivery process, thecross linking primarily occurs after the delivery process (i.e., duringthe fixation process). The delivery process can be performed to balancethe beneficial properties associated with substantially completediffusion while minimizing or limiting the effects associated withinitializing or promoting cross-linking. In some embodiments, the rateof diffusion can be maximized while limiting and minimizing anydeleterious effects associated with increased cross-linking rate.

To perform the fixation process, the tissue sample can be removed fromthe cold fixative and immersed in warm fixative to start cross-linkingand/or increase the rate of cross-linking. The temperature of the warmfixative can be greater than the ambient temperature and up to at least55° C., more typically from about 35° C. to about 45° C., as thistemperature range may increase the cross-linking kinetics sufficientlyto allow relatively quick tissue cross-linking. However, if thetemperature is increased above about 50° C., the tissue sample may beginto degrade, which may have a deleterious effect on certain subsequenthistological reactions. Thus, the upper temperature and time period ofthe fixation process can be selected to allow subsequent imagingprocesses, such as in situ hybridization, IHC, H&E, western blotting,PCR, and/or sequencing and nucleic acid analysis. The time period forthe fixation process can range from greater than about 15 minutes up toat least about 5 hours, more typically is at least about 1 hour to about4 hours, and more typically is from about 2 hours to about 3 hours. Incertain embodiments, the fixation process can be performed for about 1.5hour at a temperature of about 45° C.

The fixatives in the delivery process and the fixation process can bethe same or different. As yet another example, entirely differentaldehyde fixatives, such as formaldehyde and glutaraldehyde, can be usedfor the delivery and fixation processes. Additionally, instead ofremoving the sample from the fixative, the fixative can be heated forfixation. As such, the tissue sample can remain immersed in the samefixative throughout the diffusion and fixation processes.

Various factors may be considered to determine processing conditions fora particular tissue sample. These factors can include: sample thickness,which typically ranges from about 1 mm to about 10 mm thick, moretypically from about 2 mm to about 8 mm thick, and even more typicallyfrom about 4 mm to about 6 mm thick; volume of fixative to tissue samplemass, which typically is from about 10:1 to about 50:1 volume to mass;fixative composition; temperature; and sample immersion time in thefixative. Other factors can also be considered to determine processingconditions.

FIG. 1 shows a processing system 100 for processing specimens. Theprocessing system 100 includes a specimen holder 110, a container 140,and an analyzer 114 positioned in the container 140. The analyzer 114includes a transmitter 120 and a receiver 130. A computing device 160 iscommunicatively coupled to the analyzer 114.

FIG. 2 shows the container 140 with a chamber 180 filled with aprocessing media 170. The specimen holder 110, the transmitter 120, andthe receiver 130 are submerged in the processing media 170. A thermaldevice 162 can increase or decrease the temperature of the media 170 to,for example, perform processes at different temperatures. The thermaldevice 162 can include, without limitation, one or more refrigerationsystems, heaters (e.g., resistance heaters, electric heaters, etc.),thermoelectric devices (e.g., Peltier devices), or the like.

To presoak a tissue sample 150, the processing can be a cold fixative ata temperature in a range of about 0° C. to about 5° C. The computingdevice 160 can cause the transmitter 120 to output energy that passesthrough the specimen 150. The receiver 130 can receive the energy andcan send signals to the computing device 160 in response to the receivedenergy. The computing device 160 analyzes those signals to monitorprocessing. Once processing is complete, the specimen holder 110 can beconveniently removed from the container 140 or the processing media 170can be deactivated. After the desired level of diffusion is achieved,the thermal device 162 can increase the average temperature of the media170 to promote cross-linking. Once fixation is achieved, the sample 150can be removed from the media 170.

The specimen 150 can be one or more biological samples. A biologicalsample can be a solid tissue sample (e.g., any collection of cells)removed from a subject. In some embodiments, a biological sample ismountable on a microscope slide and includes, without limitation, asection of tissue, an organ, a tumor section, a smear, a frozen section,a cytology prep, or cell lines. An incisional biopsy, a core biopsy, anexcisional biopsy, a needle aspiration biopsy, a core needle biopsy, astereotactic biopsy, an open biopsy, or a surgical biopsy can be used toobtain the sample. A freshly removed tissue sample can be placed in theprocessing media 170 within an appropriate amount of time to prevent orlimit an appreciable amount of degradation of the sample 150. In someembodiments, the sample 150 is excised from a subject and placed in themedia 170 within a relatively short amount of time (e.g., less thanabout 2 minutes, 5 minutes, 30 minutes, 1 hour, 2 hours, or the like).Of course, the tissue sample can be fixed as soon as possible afterremoval from the subject. The specimen 150 can also be frozen orotherwise processed before fixation.

To analyze the specimen 150 using acoustic energy, the transmitter 120can output acoustic waves. The acoustic waves can be infrasound waves,audible sound waves, ultrasound waves, or combinations thereof.Propagation of the acoustic waves through the specimen 150 may changebecause of changes to the specimen 150. If the process involvesdiffusion, the acoustic properties of the specimen 150 can change as themedia 170 infuses the specimen 150. If the process involvescross-linking, mechanical properties (e.g., an elastic modulus) of thespecimen 150 may change significantly as cross-linking progressesthrough the tissue. The change in elastic modulus may alter the acousticcharacteristics of the specimen 150. Acoustic characteristics include,without limitation, sound speeds, transmission characteristics,reflectance characteristics, absorption characteristics, attenuationcharacteristics, or the like. To evaluate transmission characteristics,a time of flight of sound (e.g., audible sound, ultrasound, or both),the speed of sound, or the like can be measured. The TOF is a lengthtime that it takes for acoustic waves to travel a distance through anobject or substance. In some embodiments, the TOF is the length of timeit takes acoustic waves to travel through a specimen in comparison tothe time to travel through the medium displaced by the specimen. In someembodiments, the time of flight of the medium and the measurement device(e.g., the holder) may be recorded prior to insertion of the sample andstored for later reference so that it can be used for temperaturecompensation, evaporative losses, compensation protocols, predictivemodeling, or the like. The thickness of the specimen 150 can besufficiently large to produce a measurable change in the TOF. Inreflectance embodiments, the TOF can be the length of the time theacoustic waves travel through a portion of the tissue sample. Forexample, the TOF may be the length of time that the acoustic wavespropagate within a portion of the tissue sample. Thus, the TOF can becalculated based on acoustic waves that travel through the entirespecimen, acoustic waves reflected by the tissue sample, or both.

The speed of acoustic waves traveling through the specimen 150 isgenerally equal to the square root of a ratio of the elastic modulus (orstiffness) to the density of the specimen 150. The density of thespecimen 150 may remain generally constant and, thus, changes in thespeed of sound and the changes in TOF are primarily due to changes inthe specimen's elastic modulus. If the density of the specimen 150changes a significant amount, the sound speed changes and the TOFchanges attributable to a change in elastic modulus can be determined byconsidering the specimen's changing density. Thus, both static anddynamic characteristics of the specimen 150 can be analyzed.

The processing system 100 can be a closed loop system or an open loopsystem. In closed loop embodiments, acoustic energy is transmittedthrough the specimen 150 based upon feedback signals from the receiver130 and/or signals from one or more sensors configured to detect aparameter (e.g., temperature, pressure, or any other measurableparameter of interest) and to transmit (or send) signals indicative ofthe detected parameter. Based on those signals, the processing system100 can control operation of the transmitter 120. Alternatively, theprocessing system 100 can be an open loop system wherein the transmittedacoustic energy is set by, for example, user input. It is contemplatedthat the processing system 100 can be switched between a closed loopmode and an open loop mode.

The specimen holder 110 can be portable for conveniently transporting itbetween various locations. In a laboratory setting, a user can manuallytransport it between workstations or between equipment. The illustratedspecimen holder 110 is in the form of a cassette with a rigid main body210 that surrounds and holds the specimen 150. The main body 210includes a first plate 220 and a second plate 230 spaced apart from thefirst plate 220 to define a receiving space or chamber 240. The specimen150 is positioned in the receiving space 240. The plates 220, 230 canhave apertures or other features that facilitate transmission ofacoustic energy. The shape, size, and dimensions of the specimen holder110 can be selected based on the shape, size, and dimensions of thespecimen 150. In various embodiments, the specimen holder can be (orinclude) a cassette, a rack, a basket, a tray, a case, foil, fabric,mesh, or any other portable holder capable of holding and transportingspecimens. In some embodiments, the specimen holder 110 is a standardbiopsy cassette that allows fluid exchange.

With continued reference to FIGS. 1 and 2, the transmitter 120 and thereceiver 130 are fixedly coupled to walls 247, 249 of the container 140by brackets 250, 260, respectively. The container 140 can be a tank, atub, a reservoir, a canister, a vat, or other vessel for holding liquidsand can include temperature control devices, a lid, a covering, fluidiccomponents (e.g., valves, conduits, pumps, fluid agitators, etc.), orthe like. To pressurize the processing media 170, the chamber 180 can bea pressurizable reaction chamber. Additionally the chamber 180 can beoperated under a vacuum to reduce air bubble formation impeding soundtransmission, and to support easier perfusion of fluids into thespecimen holder 110 to displace trapped air.

To minimize, limit, or substantially eliminate signal noise, thecontainer 140 can be made, in whole or in part, of one or more energyabsorbing materials (e.g., sound absorbing materials, thermallyinsulating materials, or the like). The size and shape of the container140 can be selected to prevent or substantially eliminate unwantedconditions, such as standing waves, echoing, or other conditions thatcause signal noise. For example, if acoustic waves reflect off the innersurfaces of the container 140 and result in signal noise, the size ofthe container 140 can be increased.

The transmitter 120 can include a wide range of different types ofacoustic elements that can convert electrical energy to acoustic energywhen activated. For example, an acoustic element can be a singlepiezoelectric crystal that outputs a single waveform. Alternatively, anacoustic element may include two or more piezoelectric crystals thatcooperate to output waves having different waveforms. The acousticelements can generate acoustic waves in response to drive signals fromthe computing device 160 and can output at least one of audible soundwaves, ultrasound waves, and infrasound waves with different types ofwaveforms. The acoustic waves can have sinusoidal waveforms, stepwaveforms, pulse waveforms, square waveforms, triangular waveforms,saw-tooth waveforms, arbitrary waveforms, chirp waveforms,non-sinusoidal waveforms, ramp waveforms, burst waveforms, pulsecompression waveforms (e.g., window chirped pulse compressionwaveforms), or combinations thereof. In some embodiments, the acousticelements are transducers capable of outputting and detecting acousticenergy (e.g., reflected acoustic energy). Such embodiments are wellsuited to evaluate the specimen based on reflected acoustic waves. Forexample, the transmitter 120 can be in the form of an ultrasoundtransducer that transmits acoustic waves through at least a portion ofthe tissue sample 150. At least some of the acoustic waves can bereflected from the tissue sample 150 and received by the ultrasoundtransducer 120. A wide range of different signal processing techniques(including cross-correlation techniques, auto-correlation techniques,echoing analysis techniques, phase difference analysis, integrationtechniques, compensation schemes, synchronization techniques, etc.) canbe used to determine a TOF of the acoustic waves. The computing device160 can thus evaluate acoustic energy that is transmitted through theentire specimen 150 or acoustic energy reflected from the specimen 150,or both.

Audible sound waves may spread out in all directions, whereas ultrasoundwaves can be generally collimated and may reduce noise caused byreflectance and enhance transmission through the specimen 150. As usedherein, the term “ultrasound” generally refers to, without limitation,sound with a frequency greater than about 20,000 Hz (hertz). For a givenultrasound source (e.g., an ultrasound emitter), the higher thefrequency, the less the ultrasound signal may diverge. The frequency ofthe ultrasound signals can be increased to sufficiently collimate thesignals for effective transmission through the processing media 170 andthe specimen 150. To analyze a fragile specimen, relatively highfrequency ultrasound can be used to minimize, limit, or substantiallyprevent damage to such specimen.

Additionally or alternatively, the transmitter 120 can include, withoutlimitation, energy emitters configured to output ultrasound,radiofrequency (RF), light energy (e.g., visible light, UV light, or thelike), infrared energy, radiation, mechanical energy (e.g., vibrations),thermal energy (e.g., heat), or the like. Light emitters can be lightemitting diodes, lasers, or the like. Thermal energy emitters can be,without limitation, heaters (e.g., resistive heaters), cooling devices,or Peltier devices. Energy emitters can cooperate to simultaneously orconcurrently deliver energy to the specimen 150 to monitor a wide rangeof properties (e.g., acoustic properties, thermal properties, and/oroptical properties), to reduce processing times by keeping the media 170at a desired temperature, enhance processing consistency, combinationsthereof, or the like.

The receiver 130 can include, without limitation, one or more sensorsconfigured to detect a parameter and to transmit one or more signalsindicative of the detected parameter. The receiver 130 of FIGS. 1 and 2includes at least one sensor configured to detect the acoustic energyfrom the transmitter 120. In other embodiments, the receiver 130 caninclude one or more RF sensors, optical sensors (e.g., visible lightsensors, UV sensors, or the like), infrared sensors, radiation sensors,mechanical sensors (e.g., accelerometers), temperature sensors, or thelike. In some embodiments, the receiver 130 includes a plurality ofdifferent types of sensors. For example, one sensor can detect acousticenergy and another sensor can detect RF energy.

The computing device 160 of FIG. 1 is communicatively coupled (e.g.,electrically coupled, wirelessly coupled, capacitively coupled,inductively coupled, or the like) to the transmitter 120 and thereceiver 130. The computing device 160 can include input devices (e.g.,a touch pad, a touch screen, a keyboard, or the like), peripheraldevices, memory, controllers, processors or processing units,combinations thereof, or the like. The computing device 160 of FIG. 1 isa computer, illustrated as a laptop computer.

FIG. 3 shows the computing device 160 (illustrated in dashed line)including a signal generator 270, a processing unit 280, and a display290. The signal generator 270 can be programmed to output drive signals.Drive signals can have one or more sinusoidal waveforms, step waveforms,pulse waveforms, square waveforms, triangular waveforms, saw-toothwaveforms, arbitrary waveforms, chirp waveforms, non-sinusoidalwaveforms, ramp waveforms, burst waveforms, or combinations thereof. Thewaveform can be selected based on, for example, user input, storedparameters, or input from another system (e.g., a tissue preparationunit, staining unit, etc.). By way of example, the signal generator 270can include an arbitrary function generator capable of outputting aplurality of different waveforms. In some embodiments, the signalgenerator 270 is an arbitrary signal generator from B&K Precision Corp.or other arbitrary signal generator.

The computing device 160 is communicatively coupled to a tissueprocessing unit that applies any number of substances to prepare thespecimen for embedding. The computing device 160 can prepare a tissuepreparation protocol that is used by the tissue processing unit. Thetissue preparation protocol can include a length of processing time fora particular substance, target composition of a substance, temperatureof a particular substance, combinations thereof, or the like.

The processing unit 280 can evaluate the change in the TOF of sound inthe specimen 150 by, for example, comparing the acoustic waves outputtedby the transmitter 120 to the acoustic waves detected by the receiver130. This comparison can be repeated any number of times to monitor thefixation state of the specimen 150. In some embodiments, the processingunit 280 determines a first length of time it takes the acoustic wavesto travel through the specimen 150. The processing unit 280 thendetermines a second length of time it takes a subsequently emittedacoustic wave to travel through the specimen 150. The first length oftime is compared to the second length of time to determine, withoutlimitation, a change in speed (e.g., acceleration) of the sound waves,an absolute and/or relative change in TOF, change in distance betweenthe transmitter 120 and the receiver 130, change in temperature and/ordensity of the processing media 170, or combinations thereof. Theprocessing unit 280 can use different types of analyses, including aphase shift analysis, an acoustic wave comparison analysis, or othertypes of numerical analyses.

To store information, the computing device 160 can also include memory.Memory can include, without limitation, volatile memory, non-volatilememory, read-only memory (ROM), random access memory (RAM), and thelike. The information includes, but is not limited to, protocols, data(including databases, libraries, tables, algorithms, records, audittrails, reports, etc.), settings, or the like. Protocols include, butare not limited to, baking protocols, diffusion protocols, fixationprotocols, tissue preparation protocols, staining protocols,conditioning protocols, deparaffinization protocols, dehydrationprotocols, calibration protocols, frequency adjustment protocols,decalcification protocols, or other types of routines. Protocols thatalter or impact tissue density or sound transmission can be used tocontrol the components of the computing device 160, components of theanalyzer 114, microscope slide processing units, stainers, ovens/dryers,or the like. Data can be collected or generated by analyzing thespecimen holder 110, the processing media 170, the specimen 150, or itcan be inputted by the user.

The computing device 160 can evaluate different acoustic properties.Evaluation of acoustic properties can involve comparing sound speedcharacteristics of the specimen, comparing sound acceleration in thespecimen, analyzing stored fixation information, and analyzing TOF.Analysis of the TOF may involve, without limitation, evaluating thetotal TOF, evaluating changes in TOF over a length of time (as discussedabove), evaluating rates of change in TOF, generating TOF profiles, orthe like. The stored fixation information can include, withoutlimitation, information about sound speeds for different types oftissue, TOF of reagents, fixation rates, predicted fixation time,compensation protocols, percent cross-linking, TOF profiles, tissuecompositions, tissue dimensions, algorithms, waveforms, frequencies,combinations thereof, or the like. In some embodiments, the computingdevice 160 evaluates at least one of the TOF, a TOF change, amplitude ofthe sound waves, an intensity of the sound waves, phase shifts, echoing,a temperature and/or density of the specimen 150, and a temperatureand/or density of the processing media 170.

The computing device 160 can select, create, or modify fixationsettings, with or without prior knowledge of specimen history, specimenfixation state, or type of tissue so as to improve the reliability andaccuracy of diagnosis, especially an advanced diagnosis. Fixationsettings include, without limitation, length of fixation time (e.g.,minimum fixation time, maximum fixation time, ranges of fixation times),composition of the processing media, and temperature of the processingmedia. By way of example, if the specimen 150 has a known fixationstate, an appropriate fixation protocol can be selected based, at leastin part, on the known fixation state. If the specimen 150 has an unknownfixation state, the analyzer 114 is used to obtain information about thefixation state. For example, the analyzer 114 can obtain informationabout a specimen that is already partially or completely fixed. Protocolsettings can be selected based, at least in part, on the obtainedinformation. The protocol settings can include tissue preparationsettings, fixation protocol settings, reagent protocol settings, or thelike. In some embodiments, reagent protocol settings (e.g., types ofIHC/ISH stains, staining times, etc.) can then be selected tomatch/compensate for the fixation state based, at least in part, oninformation from the analyzer 114. The analyzer 114 can thus analyzeunfixed, partially fixed, or completely fixed specimens.

To process multiple tissue samples, the processing system 100 candynamically update fixation settings. Fixation settings can be generatedby analyzing the illustrated specimen 150. Another specimen taken fromthe same biological tissue as the specimen 150 can be processed usingthe new fixation settings. In this manner, the fixation process can bedynamically updated.

FIG. 4 shows an exemplary method of fixing the specimen 150 to protectthe specimen 150 from, for example, putrefaction, autolysis, or thelike. In general, the specimen 150 can be loaded into the processingsystem 100. The processing media 170 contacts and begins to fix thespecimen 150. The analyzer 114 monitors the fixation process. After thespecimen 150 is sufficiently fixed, the specimen 150 is taken out of thefixation media 170 to conveniently avoid under-fixation andover-fixation. Details of this fixation process are discussed below.

At step 300 of FIG. 4, the specimen 150 is loaded into the specimenholder 110. To open the specimen holder 110, the plates 220, 230 can beseparated. The plates 220, 230 can be coupled together to loosely holdthe specimen 150. In some embodiments, the specimen holder can be astandard Cellsafe™ tissue cassette for biopsy samples from Cellpath Ltdor other types compatible with acoustic transmission. The closedspecimen holder 110 is manually or automatically lowered into thecontainer 140 and held in a docking station 312 (see FIGS. 1 and 2). Thedocking station 312 can be a clamp, a gripping mechanism, or othercomponent suitable for retaining the specimen holder 110.

The processing media 170 begins to diffuse through the specimen 150 tobegin the fixation process. The fixation processes may involve limitingor arresting putrefaction, limiting or arresting autolysis, stabilizingproteins, and otherwise protecting or preserving tissue characteristics,cell structure, tissue morphology, or the like. The fixative caninclude, without limitation, aldehydes, oxidizing agents, picrates,alcohols, or mercurials, or other substance capable of preservingbiological tissues or cells. In some embodiments, the fixative is NBF.In some fixation processes, the media 170 is a fixative that causescross-linking of the specimen 150. Some fixatives may not causecross-linking.

At 310, the analyzer 114 transmits acoustic energy through the specimen150. The signal generator 270 (see FIG. 3) can output a drive signal tothe transmitter 120 which, in turn, emits acoustic energy that isultimately transmitted through the specimen 150.

At 320, the receiver 130 detects the acoustic energy and outputsreceiver signals to the computing device 160 based on the detectedacoustic energy. The receiver signals may or may not be processed (e.g.,amplified, modulated, or the like).

At 330, the computing device 160 analyzes the receiver signals. Thecomputing device 160 can control the processing system 100 to enhanceprocessing reliability, reduce processing times, improve processingquality, or the like. For example, the temperature of the processingmedia 170 of FIG. 2 can be controlled to enhance diffusion of the media170 to reduce processing times, control cross-linking, etc.

Once the tissue sample 150 reaches a desired fixation state, thespecimen holder 110 is removed (e.g., manually or automatically) fromthe media bath. The fixed specimen 150 can be embedded, sectioned, andstained without performing tests that cause specimen waste.

The processing system 100 of FIGS. 1 and 2 can include any number ofthermal devices, mechanical devices, sensors, or pumps. The mechanicaldevices can include, without limitation, agitators (e.g., fluidagitators), mixing devices, vibrators, or the like. The sensors can be,without limitation, acoustic sensors, motion sensors, chemical sensors,temperature sensors, viscosity sensors, optical sensors, flow sensors,position sensors, pressure sensors, or other types of sensors. Thesensors can be positioned at various locations about the chamber 180.

TOF measurements can be used to monitor preparation processes, includingdelivery processes, fixation processes, etc. Theoretical changes in TOFcan be calculated based on the distances between components in theprocessing system 100, the dimensions of the specimen 150, a length of asound path 313 (see FIG. 2) along which the acoustic energy travels, andthe acoustic properties of the fixative media 170 and specimen holder110. The computing device 160 can analyze calculated values to determinefixation settings, such as initial fixation settings.

Table 2 below shows calculated theoretical changes in TOF based on thespeed of sound in water (1,482 m/s), the speed of sound in unfixedmuscular tissue (1,580 m/s), and the speed of sound in fixed musculartissue (1,600 m/s). The theoretical calculations can be compared tomeasured values in order to modify the fixation process. In someembodiments, the theoretical calculations are used to determine initialsettings for the fixation process. The initial settings may includewaveforms, amplitude of acoustic energy, frequency of acoustic energy,processing temperatures, or the like.

TABLE 2 TOF after Distance TOF fixation Sound path Dimension [mm] [us][us] transmitter->cassette/specimen D1 20 29.6 specimen D2 4 6.32 6.4specimen -> specimen holder D3 1 1.48 Specimen holder ->receiver D4 2537 TOTALS 50 74.4 74.48 delta [ns] 80

FIG. 2 shows the distance D₁ from the transmitter 120 to the specimen150, the distance D₂ between opposing surfaces of the specimen 150, thedistance D₃ from the specimen 150 to the outer surface of the secondplate 230, and the distance D₄ from the specimen holder 110 to thereceiver 130. The sound speeds and densities of common tissue types arewell known in the art. These known values can be used to calculate thechange in TOF and determine initial fixation settings. Because soundspeeds are dependent on the temperature of the medium and the distanceof the measurement channel may be dependent on thermal expansioncoefficients of the related components, a reference TOF measurement ofthe medium and the measurement channel at a given temperature of themedium and the test environment may be performed in some embodiments.This reference measurement may be used to compensate for the TOFmeasurements of the specimen.

The total TOF can be determined by the individual travel times of thesound waves traveling first through a portion of the media 170 for thetime “t1” across the distance D₁, followed by the time “t2” as the soundwaves travel across the distance D₂, and finally the time “t3” as thesound waves travel the remaining distances D₃ and D₄. Thus, the totalTOF=t1+t2+t3. Changes in the total TOF can be measured and related tothe state of fixation and thus relate primarily or only to the time“t2.” The information of an unimpeded sound path (e.g., a sound pathwithout a sample insertion as a reference) may be used to identifyvariation of the total TOF due to, for example, changes of the media 170(e.g., temperature changes, density changes, etc.).

Different types of tissue can have different acoustic characteristics.FIG. 5 is a non-limiting exemplary graph of fixation phase versus changein a TOF. A curve 340 can be generated by analyzing a specimen.Different types of tissue may generate different types of curves, asdiscussed below in connection with FIGS. 30-38. The computing device 160can correlate the different curves to different tissue types. To processa fresh specimen, a curve can be selected corresponding to the same orsimilar tissue type as the fresh specimen. The computing device 160 canprovide an appropriate processing protocol based on the curve. Theprotocol can include, without limitation, a reagent delivery protocol,fixating protocol, a tissue preparation protocol, an embedding protocol,a decalcification protocol, a staining protocol, or combinationsthereof. Information can also be obtained while the protocol isperformed to modify the protocol or select another protocol. By way ofexample, the curve 340 can be used to determine, at least in part, whento remove a specimen from a fixation media.

Curve fitting techniques using polynomials, trigonometric functions,logarithmic functions, exponential functions, interpolations (e.g.,spline interpolations) and combinations thereof can be used to generatethe curve 340 which approximates collected data. Some non-limitingexemplary curve fitting techniques are discussed in connection withFIGS. 13-16.

At an initial fixation phase FP° in FIG. 5, the unfixed specimen 150 isexposed to the processing media 170. It is believed that the outermostportions of the specimen 150 may begin to cross-link and the media 170can diffuse into the specimen 150. As the fixation phase increases fromFP° to FP₁, the change in TOF gradually increases with respect to thefixation phase. From FP₁ to FP₂, the cross-linking approaches theinterior regions of the specimen 150. The change in TOF is nonlinearwith respect to the fixation phase. As the fixation phase approachesFP₂, the rate of change of the TOF change begins to rapidly decrease.From FP₂ to FP₃, the specimen 150 becomes saturated until there may beover-saturation at about FP₃. Approaching FP₃, the slope of the curve340 continues to decrease as it approaches zero, corresponding to whenthe specimen 150 may be at risk of over-fixation. The fixation processcan be controlled based on, for example, the slope of the curve 340, aminimum TOF change, a maximum TOF change, combinations thereof, or thelike.

A predictive algorithm can be used to determine a desired processingtime to achieve a desired level of fixation. The computing system 160can store and select predictive algorithms based on the desired amountof infusion, cross-linking, etc. If the fixation media 170 diffuses at anon-linear rate, a non-linear diffusion predictive algorithm can beselected. If the fixation media 170 causes cross-linking at a non-linearrate, a non-linear fixation predictive algorithm can be selected. Forexample, cross-linking could exhibit exponential decay so an exponentialdecay curve can be used to estimate an end of processing time. Thedesired level of cross-linking can be selected based on the tissue type,the analysis to be performed, the expected storage time, or othercriteria known in the art. For example, the predictive curve can be usedto determine a predicted stopping time for which cross-linking should beabout 99% complete.

A Levenberg-Marquardt algorithm or other type of nonlinear algorithm canbe used to generate an appropriate best fit curve. In some predictiveprotocols, the Levenberg-Marquardt algorithm uses an initial value togenerate a curve. A damping-undamping scheme can produce the nextiteration. Non-limiting exemplary damping-undamping schemes aredescribed in the paper “Damping-Undamping Strategies for theLevenberg-Marquardt Nonlinear Least-Squares Method” by Michael Lampton.The closer to the actual curve of the initial value, the more likely itis that the algorithm will provide the desired best-fit curve. In someprotocols, a plurality of values in the data set (e.g., a first value, amiddle value, and a last value) are used to produce an exponential curvethat fits the three values. The initial values can be selected based onknown values for similar tissue samples. After performing the iterativeprocess, a best fit curve is generated. The best-fit curve can be usedto determine the predicted state of the specimen at different timesduring processing. This can be helpful to develop a schedule to increaseprocessing throughput, especially if the processing system allows forindividual processing, as discussed in connection with FIG. 21.

FIG. 6 shows a timing relationship between a signal 360 from thetransmitter 120 and a detected signal 380. The signal 360 can have asufficient number of signal bursts to evaluate phase changes of wavesentering and exiting the specimen 150 at a particular distance. By wayof example, acoustic waves 370 of the signal 360 are illustrated as apulse burst and can be a 1 MHz sine burst with 53 cycles, 5.3 msrepetition rate, and a 7.4 V amplitude. Other acoustic waves withdifferent pulse bursts, numbers of cycles, repetition rates, amplitudes,etc. can also be used. The detected signal 380 corresponds to the signalreceived by the receiver 130. A pulse burst 390 corresponds to thesignal burst 370.

FIGS. 7 and 8 show the relationship between the signal burst 370 and thereceived acoustic waves 390. A change in TOF, if any, can be determinedbased on a comparison of the waves 370, 390. If the TOF does not change,there will be no phase shift between the waves 370, 390 over time. Ifthere is a TOF change, there will be a phase shift over time. Forexample, at the fifth wave 392, there is about 38.28 μs phase delay orshift, measured against the reference signal 370. As a sample undergoesfixation, the sound speed in most types of tissue (e.g., muscle tissues,connective tissues, etc.) typically increases. However, some fattytissues will cause a decrease of sound speed during fixation. The system100 can detect a relative phase angle difference resulting from a phaseshift caused by an early or late arrival of the pulse packet 390. Alarge number of acquisitions can be obtained. For example, about 100 toabout 1,000 phase comparisons can be performed at a rate of about 70times per second. In one embodiment, the scheme can monitor targetchanges of at least 125 ns at a frequency of about, for example, 4 MHz.

FIG. 9 shows the relationship of outputted waves 393, received waves394, and a comparison curve 391. The comparison curve 391 shows phasedifferences, illustrated as an analog voltage output, that reflect anintegrated phase difference accumulated from a comparison (e.g., asynchronous comparison) of two wave packets 395, 397. The integratedphase difference can be used to determine when to evaluate a phasedifference between the two waves 393, 394 or what part of the waves 393,394 to compare.

A trigger point, indicated by a dashed line 398, can be communicated tothe computing device (e.g., a data capture system). The trigger point398 can be selected based on a settling point, rate of change, or thelike of the curve 394. An electronic data capture system of the system160 can analyze the waves 393, 394 at the trigger point and can have aresolution around 1 ns or better (based on +/−1 sd at n=7 captured pulsepackets) in shadowed transmission mode. Any number of trigger points canbe selected along the curve 391 based on the desired amount of sampling.

FIGS. 10A and 10B show phase angle relationships based on the frequencyof outgoing waves. In FIG. 10A, an outgoing burst signal 395 a, areceived burst signal 397 a, and an initial phase relationship Ø1 causedby the signal 397 a traveling through the sample 150. FIG. 10B shows anoutgoing burst wave 395 b outputted at a frequency 2 higher than thefrequency 1 of wave 395 a of FIG. 10A. The outgoing wave 395 b of FIG.10B has a reduced wavelength as compared to the outputted wave 395 a. Assuch, the phase relationship Ø1 is different from the phase relationshipØ2. Because the TOF is primarily or only dependent on the distance oftravel and the density of the media or sample, the phase relationshipcan be freely configured by selecting the frequency (or othercharacteristics) of the outgoing waves. Accordingly, the computingsystem 160 can select the frequency of the outgoing wave based on adesired phase relationship.

Frequencies and the resulting phase relationships can be correlated todetermine how changes of the outgoing frequency will result in phaserelationship changes, which in turn can be used to monitor the sample150. A monitoring protocol can include, without limitation, outputting aplurality of waves with different frequencies to generate a plurality ofphase relationships. A comparison (e.g., an extended phase rangecomparison) can be accomplished by adaptively monitoring phase angleprogression. Outgoing frequencies can be changed (e.g., incrementallychanged) by the signal generator 270 to keep the phase relationship inthe favorable range. The phase angle change is linearly dependent on thefrequency change and therefore can be added successively as an absoluteTOF increment to any additional changes observed by the phase comparisonitself. Because most reactions being monitored are in a time range ofseveral tens of minutes, an adaptive frequency change can be easilyachieved.

The base wavelength for an ultrasound transducer may result in a phasedetection limit. For example, the ultrasound transducer 120 may output asignal at a frequency of about 4 MHz, 0-180 degrees, at about the 125 nsrange. Different ultrasound receivers may provide a larger phase anglerange, but depth resolution for the target thickness may be limited tothicker samples. For greater phase angle differences (e.g., greater than180 degrees), the integrated voltage can be reversed in polarity, orrepeat itself for phase angle differences greater than 360 degrees.Because monitoring of fixation may rely on relative phase angle changes,the initial phase angle can be optimized on a target, such as by varyingthe base wavelength in the arbitrary function generator 170 to establishan initial setting with a favorable phase relationship, for example, thepoint 399 in the graph of FIG. 11. FIG. 11 also shows phase differencesthat provide high sensitivity. Other methods may not rely on phasecomparison measurements and instead utilize chirped pulse excitation andcorrelation or convolution methods to calculate absolute TOF withsimilar precision and resolution.

A wide range of compensation techniques can be utilized to analyze TOFmeasurements. One compensation technique for relatively large phaseshifts during TOF monitoring relies on reduction principles. Amathematical reduction principle can use, for example, multiple discreteexcitation frequencies (=wavelength scans) sent in succession of burstsat the same target location. A change in time, ΔT, can represent theactual time delay between when a wave is sent and when the wave isreceived. A plurality of waves of different wavelengths, λ₁, λ₂, . . . ,λ_(n), can be emitted.

The received waves can be compared with the outgoing waves to determinecorresponding phase changes, Δθ_(λ) ₁ , Δθ_(λ) ₂ , . . . Δθ_(λ) _(n) .The computing device 160 can narrow down the actual value of ΔT to asubset of values which is much smaller than the set of all possiblevalues for ΔT. If there is a range of wavelength scans, λ₁, λ₂, . . . ,λ_(n), and their corresponding phase changes, Δθ_(λ) ₁ , Δθ_(λ) ₂ , . .. Δθ_(λ) _(n) , the computing device 160 can use each reading to furthernarrow down an estimated until there is only one feasible value ΔT,which can correspond to the absolute time-of-flight. Phase detection canbe performed using a demodulator and a controller to provide highresolution. For example, sub-nanosecond resolution at a total TOF ofabout 20 μs can be achieved with a microcontroller (e.g., an 8-bitmicrocontroller) and a demodulator chip).

FIG. 11A is a plot of frequency versus phase comparison in accordancewith one embodiment. A programmable function generator can generate manysine waves at different frequencies. By measuring the phase differenceat each frequency, we can reconstruct the actual time-of-flight. Acomparison can be performed as a function of frequency to unique time offlights. As shown in FIG. 11A, the comparison wave can be a generallytriangle wave with a slope corresponding to the time-of-flight. TOF canalso be determined based on techniques disclosed in U.S. applicationSer. No. 13/372,040, filed on Feb. 13, 2012, and incorporated byreference in its entirety.

The change in phase Δθ_(λ) can be measured at a given frequency, λ.Because there may be many values for ΔT that would yield the same Δθ₂,the ΔT can be estimated or predicted based, at least in part, on aspecific Δθ_(λ) since most values for ΔT would not yield a given Δθ_(λ)(the true value of ΔT satisfies the equation ΔT =N/(2λ)+Δθ_(λ) for someinteger N). A program can be used to at least narrow down the true valueof ΔT to a subset of values which is much smaller than the set of allpossible values for ΔT based on an estimated ΔT from a specific Δθ_(λ).The computing device 160 can generate a range of wavelength scans, λ₁,λ₂, . . . , λ_(n), and their corresponding phase changes, Δθ_(λ) ₁ ,Δθ_(λ) ₂ , . . . Δθ_(λ) _(n) as detailed above.

An interactive algorithm can be used to determine ΔT and can be used tominimize or avoid problems associated with solving for ΔT algebraically(e.g., problems attributable to the noisy nature of TOF measurements).In some interactive algorithms, a ΔT is estimated or predicted. Atheoretical Δθ_(λ) can be determined for that ΔT and can be compared tomeasured Δθ_(λ)'s to assign a penalty function. The penalty function canbe the sum of the squared differences between the theoretical Δθ_(λ)'sand the measured Δθ_(λ)'s. The true value of ΔT can be the minimizer ofthe penalty function. The method for minimizing this function can bedetermined using different techniques, such as a sweep of values or abinary search. Additionally or alternatively, a gradient descent, Newtonmethod (including Gauss-Newton algorithm), or Levenberg-Marquardt methodcould be used. Other algorithms can also be utilized, if needed ordesired. In some protocols, one or more out-of-range values (e.g.,values <0.2 and/or values >1.5) can be discarded. The out-of-rangevalues can be selected based on criteria corresponding tocharacteristics of the tissue sample.

A phase detection algorithm can be used to compare an outgoing wave witha corresponding received wave. One type of phase detection algorithm isa range extension algorithm involving multiple wavelengths of phaseangle changes for acoustic speed measurements. When the speed ofacoustic waves changes significantly, the computing device 160 may basewave comparisons on a different period of the wave than it started on,resulting in a sudden change from an increasing TOF to a decreasing TOFor resulting in a sudden change from a decreasing TOF to an increasingTOF. The sudden change is attributable to the comparison of differentphases, thereby leading to artificial data. Rate of changes in TOF canbe evaluated to determine whether the TOF changes are artificial changesdue to such out of phase comparisons. For example, the second derivativeof the TOF curve can be used to determine whether a local maximum TOF ora local minimum in TOF is a natural change in TOF or an artificialchange in TOF.

FIG. 12 shows a graph of time versus TOF signal. A curve 405 graduallydecreases at 408 to a local minimum 409. The curve 405 then increases at410 to an artificial local maximum 411. The actual TOF continues togradually increase, as indicated by the dashed curve 412. The peak 411is generated based on an out of phase comparison. The curve 405 at 413continues to decrease at time greater than 260 based on the out of phasecomparison. As shown in FIG. 12, there is a significant differencebetween the actual TOF 412 and the artificial TOF 413.

Artificial measurements can be identified to avoid the peak 411. By wayof example, FIG. 13 is a graph of time versus TOF with noisy data. TheTOF increases from a time=0 minutes to about t=55 minutes. The TOFgradually decreases from 55 minutes to about 150 minutes. The TOFsuddenly begins to increase at about 150 minutes. A program candetermine whether the sudden change in TOF is accurate or artificial.

FIG. 14 shows a plot generated using numerical differentiation (e.g.,finite-difference methods) of the data of FIG. 13 which increase thesignal-to-noise ratio resulting in jaggedness curve 414 that is notsuitable for determining whether changes in TOF are natural orartificial. The spike at time of about 155 corresponds to artificialchanges from a decreasing TOF to an increasing TOF based on a comparisonbetween difference phases of waves. Based on the numerous large spikesin FIG. 14, it may be difficult to accurately determine whether a spikecorresponds to an artificial or a natural change in TOF.

FIG. 15 shows jaggedness of the noisy two-peak curve of FIG. 13 using asmoothing algorithm, such as a total variation smoother algorithm. Thetotal variation smoothing algorithm can be used to smooth the raw dataof FIG. 13 before generating the jaggedness plot. A compensation programcan recognize that the change in time of flight at about t=150 isartificial and recompare different phases of the waves to ensure thatthe general trend of the time of flight as t approaches 150 is generallymaintained. The large spike 416 at 150 minutes can be convenientlyidentified in FIG. 15, while the natural peak is barely identifiable. Acompensation program can be used to compensate for the spike 416. Suchcompensation programs can include, without limitation, an algebraicalgorithm or other type of compensation algorithm.

Noise can be reduced without eliminating desired data. Onenoise-reducing method that does not over-smooth data (e.g., cusps) isdiscussed in “Numerical Differentiation of Noisy, Nonsmooth Data” byRick Chartrand, published by Los Alamos National Laboratory, Dec. 13,2005. The method is an example of a total variation smoother whichsmoothes noise while preserving true cusps, thereby minimizing,eliminating, or limiting only unwanted noise. FIG. 16 shows a data plot417, a first smoothed curve 418 (shown in dashed line), and a secondsmoothed curve 419. Numerous cusps of the data plot 417 are eliminatedin the first smoothed curve 418 which was generated using a numericaldifferentiation algorithm designed to remove sharp peaks/valleys. Thesecond curve 419 was generated using total variation smoother algorithmwhich preserves true cusps. Thus, the second curve 419 is well suitedfor identifying inaccurate (e.g., artificial peaks/valleys) TOF signalsat the time of 49 minutes as compared to the first smoothed curve 418.

Movement of tissue within a specimen holder can lead to inaccuratemeasurements. If the tissue sample 150 moves inside the specimen holder110, the change in position of the specimen can significantly altermeasurements for monitoring cross-linking, changes in specimen density,or the like. Averaging, comparing, or otherwise analyzing data obtainedfrom one or more analyzers, as discussed in connection with FIG. 18, canbe used to compensate for such movement. The computing device 160, forexample, can include different types of algorithms that use dataobtained for a plurality of analyzers. If tissue shifts within thespecimen holder (e.g., when a cassette is jarred or a cassette movesrapidly through media), movement of the tissue relative to the cassettecan be accounted for to avoid changes attributable to tissue migration.

Tissue analyzers described herein can also analyze tissue samples afterfixation. For example, the tissue analyzer 114 of FIGS. 1 and 2, or amodified tissue analyzer, can obtain information about a tissue sampleembedded in a material, a cut mountable section (e.g., a cut strip ofembedded tissue), or the like. Information about the specimen can thusbe obtained before fixation/processing, during fixation/processing, andafter fixation/processing. Specimens can be analyzed any number of timesthroughout processing to ensure that the specimen is properly preparedfor examination. One method of analyzing fixed tissue is described belowwith respect to an embedded specimen.

In some embodiments, the specimen 150 is a block of embedding materialcontaining a tissue sample. The embedding material can have mechanicalproperties that may facilitate sectioning. Materials for embeddinginclude, but are not limited to, paraffin, resin (e.g., plastic resins),polymers, agarose, nitrocellulose, gelatin, mixtures thereof, or thelike. Paraffin is a white or generally colorless water insoluble solidsubstance that is resistant to many reagents. Paraffin can be a mixtureof hydrocarbons chiefly of the alkaline series obtained from petroleum.A wide range of different mixtures of similar hydrocarbons can be usedto make paraffin, and these mixtures can be solid, semi-solid, and/oroily. The acoustic properties of these types of embedding materials maybe known or may be determined using the analyzer 114. The speed of soundtraveling through the block (including the tissue) can be analyzed toselect an appropriate protocol to be performed on the tissue sample. Awide range of different variables (e.g., dimensions of the block, degreeof fixation of the tissue, temperature of the block, temperature of thetissue, etc.) can affect the speed of sound. Although the density of theembedding material may impact sound speeds, TOF measurements may yieldimportant information about tissue properties, tissue fixation state,the impregnating process used to embed the tissue, or the like. Thecontribution to the sound speed by the tissue can be isolated out fromthe contribution to the sound speed of the embedding material toevaluate the properties of the tissue.

After analysis, the embedded specimen can be cut into mountablesections, placed on a microscope slide, and then dried. A microtome cancut the specimen into thin mountable sections, for example, slices onthe order of about 5 microns to about 6 microns thick. Each section caninclude a portion of the tissue sample and some of the embeddingmaterial. Different techniques can be used to transfer the tissuesamples onto the microscope slide. In some embodiments, the cut sectionsare floated on water to spread or flatten the sections. If the sectionsare pieces of paraffin embedded tissue, the sections can be floated on awarm bath to keep the sections in generally flat configurations, therebyreducing or preventing folding, creasing, or bending. A microscope slideis inserted into the warm bath. A front surface of the slide is used topick up the tissue samples.

Reagents can be applied to the tissue samples. The composition of thereagent, processing times, or volume of reagent can be selected based onthe information obtained by the processing system 100. Stainingprotocols for the embedded tissue samples can be selected with limitedor substantially no known information about the tissue sample 150. Evenarchived tissue samples can be matched with suitable reagents. Reagentsinclude, without limitation, stains, wetting agents, probes, antibodies(e.g., monoclonal antibodies, polyclonal antibodies, etc.), antigenrecovering fluids (e.g., aqueous- or non-aqueous based antigen retrievalsolutions, antigen recovering buffers, etc.), or the like. Stainsinclude, without limitation, dyes, hematoxylin stains, eosin stains,conjugates of antibodies or nucleic acids with detectable labels such ashaptens, enzymes or fluorescent moieties, or other types of substancesfor imparting color and/or for enhancing contrast.

The analyzer 114 can be used to determine whether the specimen 150 hasbeen fixed and, if so, the degree of fixation. If the specimen 150 hasnot been fixed, the specimen 150 can be fixed. If the specimen 150 isproperly fixed, the specimen 150 can be removed from the fixative bathor the fixative can be deactivated. Deactivation of the fixative 170 canbe achieved by diluting the fixative, exchanging fluids, rendering thefixative inactive, or the like.

If the specimen 150 has been left in the fixative 170 for an extendedperiod of time, it may be over-fixed. Specimens are often inadvertentlyleft in fixatives, for example, overnight. In such cases, the specimenmay not need any additional fixing. The analyzer 114 can analyze thecharacteristic sound speeds of the specimen 150 and compare the measuredcharacteristic sound speed to a typical sound speed for the tissue typeof the specimen 150. Based on the comparison, the computing device 160can determine the degree of fixation, if any, of the specimen 150. Forexample, if the sound speed does not change a threshold amount within anexpected time frame, the specimen 150 is already fixed. Thus, thespecimen 150 can be removed from the fixation bath or the fixationprocess can be stopped to avoid over-fixation. By way of anotherexample, the measured characteristics can be compared to stored values(e.g., sound speed characteristics of fixed tissue) to determine thedegree of fixation. If the specimen 150 is already fixed, thecharacteristic sound speed will correspond to the sound speed of fixedtissue.

FIG. 17 shows a processing system 420 with a computing device 426configured to perform signal comparison by, for example, capturing andanalyzing ultrasound phase velocity changes. The computing device 426can monitor perfusion, thermal equilibration, alcohol contraction,evaporation, fixation, combinations thereof, or the like. The computingdevice 420 is similar to the computing device 160 discussed inconnection with FIG. 3, except as detailed below.

A function generator 421 can send signals to a synchronization device423 and to a transmitter 429. A controller 422 sends signals to thesynchronization device 423 and to a positioning mechanism 430. Thepositioning mechanism 430 positions a sample between the transmitter 429and a receiver 431 based, at least in part, on the signals from thecontroller 422.

The synchronization device 423 can synchronize signals based on phaseshifts, outputted/received frequencies, signal comparisons, or the likeand outputs signals to a capture system 424. The capture system 424 canbe a data capture system the relies on an internal or external clock. Insome embodiments, the capture system 424 can be an Omega DAC 3000 soldby Omega Engineering, Inc. or similar type of device. Other types ofcapture systems can also be utilized, if needed or desired.

A signal conditioner 425 receives output from the function generator 421and output from the receiver 431. An analog or digital phase/ratiocomparator 432 outputs signals to an integrator 433 (e.g., a digitalintegrator, an analog integrator, etc.), which in turn outputs signalsto the capture system 424. The signal conditioner 425 can include othercomponents, circuits, signal processing units such as DSPs, FPGAs,digital-to-analog devices, analog-to-digital devices, amplifiers (e.g.,gain amplifiers), RF/IF gain phase detectors, or the like.

A computing unit 434 receives signals from the capture system 424 andcan include frequency/phase shift databases for correlating phase shiftsor convolutions for chirped pulse excitation to fixation states, controlmaps, fixation data, protocols, or the like. The computing unit 434 cancontrol the components of the computing system 420. By way of example,the function generator 421 and the controller 422 can be controlled toautomatically monitor and process the specimen 435.

The system 420 can perform ultrasound velocity measurements based onphase differences observed between transmitted pulse packet (e.g.,100-300 waves of constant wavelength) and received pulse packet afterexposure to the sample tissue 435. The phase differences can be measuredas an absolute phase angle difference relative to the wavelength of thebase frequency of the pulse packet (e.g., 0 degrees to 360 degrees).

FIG. 18 shows a processing system 450 that includes a transportapparatus 460 configured to successively move specimen holders 470 a,470 b, 470 c, 470 d (collectively 470) to an analyzer 480. The transportapparatus 460 includes arms 520 a, 520 b, 520 c, 520 d (collectively520) that extend outwardly from a member 490. The specimen holders 470are carried by the respective arms 520. The specimen holder 470 a isshown in the analyzer 480. To move the specimen holder 470 d into theanalyzer 480, the member 490 is rotated (e.g., in a clockwise directionindicated by arrows 496) about an axis of rotation 498 until thespecimen holder 470 d is between a transmitter 500 and a receiver 502 ofthe analyzer 480. A positioning mechanism in the form of a drive motor504 can rotate the member 490 based on feed back from the analyzer 480.A computing device, for example, can control the motor 504 in responseto signals from the analyzer 480. The motor 504 can be a drive motor,stepper motor, or the like.

A fixative (not shown in FIG. 18) held in a container 488 can fix thespecimens in the specimen holders 470. Advantageously, when a pathbetween the transmitter 500 and the receiver 502 is unobstructed, theacoustic characteristics of the media can be evaluated to determine anychanges in sound speed due to the media. The processing system 100 canthen be recalibrated. If the distance between the transmitter 500 andthe receiver 502 is about 50 millimeters, signals can be sent every fewmilliseconds because the total travel time may be about 40 μs. Thefrequency of transmitted acoustic energy, focal properties oftransmitters, and geometry and dimensions of the transmitters can beselected to achieve a desired total travel time. Any number of signalscan be sent at regular or irregular intervals to determine anyprocessing changes that may affect the collected data.

The specimens can be individually monitored while all of the specimensundergo fixation. The processing system 450 can also have any number ofanalyzers 480. For example, the processing system 450 can have analyzersthat are spaced apart from each other such that the specimen holders 470are successively delivered to the analyzers. The analyzers may havedifferent types of components to evaluate different properties of thespecimens.

FIG. 19 shows a processing system 560 for automatically processingspecimens in different fluids. The samples can be processed in batchessuch that each batch of specimens is processed using the same protocol.The system 560 includes a drive apparatus 570 with a rail 580 and atransport apparatus 586 movable along the rail 580. The transportapparatus 586 includes a vertically movable rod 588 connected to acarriage 590. The carriage 590 can slide along the rail 580 to move thetransport apparatus 586 between containers 592 a, 592 b.

To move the illustrated transport apparatus 586 to the container 592 b,the carriage 590 raises the transport apparatus 586 from a loweredposition 593, as indicated by an arrow 594. Once the transport apparatus586 is out of the container 592 a, the carriage 590 can move along therail 580, as indicated by arrows 595. Once the raised transportapparatus 586 is above the container 592 b, the carriage 590 lowers thetransport apparatus 586 into the container 592 b. In this manner,specimen holders carried by the transport apparatus 586 can be submergedin processing media in the containers 592 a, 592 b. In some embodiments,including the illustrated embodiment, the container 592 a contains afixative, and the processing media in the container 592 b is a clearingagent.

Any number of containers can be used with the illustrated processingsystem 560. FIG. 20 shows a modified embodiment of the processing system560 with containers 592 a, 592 b, 592 c, 592 d (collectively 592). Acarriage 590 can carry the specimens sequentially into the containers592, which can contain a wide range of different types of processingmedias, including fixatives, clearing agents (e.g., xyline or the like),infiltrations, dehydration agents, reagents, or the like. Theillustrated processing system 560 includes a tissue preparation unit 597comprising the containers 592 b, 592 c, 592 d.

The container 592 a can contain a fixative in which specimens are fixed.After fixing, the specimens can be sequentially delivered to thecontainers 592 b, 592 c, 592 d which each contain a tissue preparationmedia, such as a dehydration agent, a clearing agent, an infiltrationagent, or the like. In some embodiments, the computing device 160 cangenerate a tissue preparation protocol used to process the specimens inthe container 592 b, which contains a dehydration agent, such asalcohol. The tissue sample can be treated with a clearing agent in thecontainer 592 c. The specimen can be treated with an infiltration agentin the container 592 d. The tissue preparation protocol can includelength of processing times in the processing media, composition of theprocessing media, temperature of the processing media, or the like. Ofcourse, different specimens with different types of tissue, dimensions,etc. can be processed for different lengths of time. As such, differenttissue preparation protocols can be generated for different tissue typesto ensure that the specimens are adequately prepared for embedding.

FIG. 21 shows a processing system 550 for automatically processingspecimens and random access loading. STAT processing can reduce the timeto diagnosis for high priority samples. The processing system 550includes a drive apparatus 552 with a rail 553 and a handing device,illustrated as a 3-axis handling robot 554, movable along the rail 553.The 3-axis handling robot 554 includes a lifter 556 for transportingspecimen holders to stations, illustrated as containers 555 a, 555 b,555 c (collectively 555). Specimen holders can be loaded at any timefrom a feed mechanism 557 a (illustrated in dashed line) to thecontainer 555 a. Each container 555 includes a rotary positioningmechanism 561 for sequentially positioning specimens in a channel of ananalyzer 559. The specimens can be monitored to ensure proper fixation.New samples can be automatically loaded at any time. In contrast to thebatch operations discussed in connection with FIGS. 18-20, once aspecimen is processed, it can be removed from the container while otherspecimens are processed.

Specimen holders can be placed in the first processing container 555 aholding cold formalin or warm formalin. The specimens can besequentially fed through a measurement channel (e.g., an ultrasound TOFmeasurement channel) in order to track the progression of the reactionin the container 555 a. Once a tissue sample process is complete, thehandling robot 554 can remove the specimen holder with the processedsample and move it to the next processing container or station 555 b.The individual handling of specimen holders can allow faster fixing ofsamples to be moved earlier and bypassing of slower fixing samples,thereby providing custom processing times optimized per individualsample. This increases the total throughput of the system 550.

By way of example, one specimen holder carrying a fatty tissue can beprocessed using a fatty tissue preparation protocol and another specimenholder carrying muscle tissue can be processed using a muscle tissuepreparation protocol. The different protocols can provide differentprocessing times, different waves (e.g., different frequencies,different waveforms, etc.), different compensation algorithms, or thelike. A protocol can be selected based on individual sample treatmentrequirements due to size variations, type of tissue sample, history oftissue samples, and/or other characteristics of samples. If thesize/material of sample changes, another protocol can be selected by theoperator and/or automatically selected by a computing system.

Information collected from samples can be used for processing subsequentsamples. Processing time information obtained from a sample can be usedto determine a priori the processing times for the next station ormonitoring (e.g., ultrasound TOF monitoring). The specimen holder caninclude information (e.g., machine-readable code) readably by readers atthe containers 555 a-c. Once processing is complete, the samples may beinfused with paraffin and can be unloaded into an output queue 557 b.The processed specimens can be picked up at convenient times.

FIG. 22 shows a processing system 556 that provides random access totissue samples. The processing system 556 includes a carouselpositioning mechanism 557. A lifter 558 can grip and carry specimenholders or specimens between processing stations 558 a-j, illustrated asopen containers.

FIG. 23 shows a station 562 with a single closed reaction chamber 563and a positioning system in the form of a mechanical drive mechanism564. A pump 566 can exchange media. A valve/multiplexer system 567fluidically couples containers 567 a-d. Additionally or alternatively,one or more vacuum devices can be used to transport fluids betweencontainers. Any number of multiplexer pumps, valve systems, vacuumdevices, conduits, thermal devices, containers, or other fluid devicescan be used to manage processing media.

FIG. 24 is a flow chart of a workflow system 568. Generally, theworkflow system 568 is used to track samples with the correspondingsubject identification from surgery through tissue processing.Information obtained from the samples can be included in the subject'srecords and can facilitate generation of reports (e.g., reports used fordiagnosis, patient monitoring, billing, etc.), an audit trail (e.g., anaudit trail of specimen handling steps), a processing parameter log(e.g., a log that could be printed and as a quality record at the end ofthe processing), or the like.

Samples can be monitored by an active (or passive) RFID tag embedded inor otherwise coupled to the specimen holder. Once the sample isacquired, it can be transferred to the specific specimen holder. In someprotocols, the specimen can be stored in a cooled container (−4° C.)with 10% neutral buffered formalin by volume. Upon entry into thespecimen holder and container, the RFID tag can be programmed, such asby swiping past a communication device (e.g., reader/writer device) totrack the time and allow association of the patient ID to the uniqueRFID device ID. Alternatively, a bar coding scheme with a linkeddatabase or other machine-readable code could be used, if needed ordesired.

At 569, a sample is taken from a subject. The sample can be a tissuesample removed from a subject using a needle, biopsy tool, or the likeand can be a section of tissue, an organ, a tumor section, a smear, afrozen section, a cytology prep, or cell lines. An incisional biopsy, acore biopsy, an excisional biopsy, a needle aspiration biopsy, a coreneedle biopsy, a stereotactic biopsy, an open biopsy, or a surgicalbiopsy can also be used to obtain the sample.

At 571, the sample is loaded into a specimen holder withmachine-readable code. The machine-readable code can be any type ofoptical symbology, magnetic pattern or electromagnetic or electrostaticsignal having information content. For example, information content mayrelate to sample identity, sample origin, sample chain of custody,instructions for processing a sample, information regarding thecharacteristics of a sample, test results for a sample, images of thesample and the like.

The workflow system 568 can include any number of communication devicescapable of reading and/or writing information. A communication devicecan be any type of machine that can decipher, translate or interpret theinformation contained in a machine-readable code, for example, a devicethat converts the code into commands for performing an automatedprocedure or presenting the information in a human-readable orhuman-interpretable form. A communication device can be a readercompatible with one or more different types of machine-readable code,such as optical symbologies, bar codes, and the like. Examples ofoptical symbologies include characters, bar codes and dataglyphs.Particular examples of bar codes include linear bar codes,multi-dimensional bar codes such as 2D stacked symbologies and 2D matrixsymbologies, and composite bar codes such as reduced-space symbologies.Even more particular examples of 2D optical symbologies include PDF417,data matrix, maxicode, vericode, codablock, aztec code, code 16K and QRcode. Bar code readers for these and any number of other opticalsymbologies are well known. Where the machine-readable code comprisescharacters (e.g., alphanumeric characters such as English text andArabic numbers) the code reader can be an optical character reader(OCR). Magnetic stripes are only one example of a device that can storeinformation in the form of a magnetic pattern. An example of anelectromagnetic code is an RFID tag. RFID tags typically include a smallmetallic antenna and a silicon chip, and can be active or passive. RFIDcode readers are well known, and typically include an antenna and atransceiver that receives information from the RFID tag. The informationcontent of an RFID tag can be fixed or changeable. In anotherembodiment, the communication device is a code reader that includes aCCD camera and the CCD camera can be used for simultaneous detection ofsamples and reading of a bar code or characters. Other examples ofmachine-readable codes that can be used include Bragg-diffractiongratings and micro- or nano-bar codes (such as spatial and spectralpatterns of fluorescent particles or spatial patterns of magneticparticles).

At 573, the sample can be pretreated to facilitate subsequentprocessing. The sample can be pre-treated with formalin or other media.Cold formalin can pre-treat the sample without causing appreciablecross-linking. The pre-treatment process can include a delivery processin which the formalin travels through the sample and are discussed inconnection with FIGS. 39-42.

At 575, the sample can be delivered to a processing system and undergoesa fixation process. The sample can be monitored during fixation.Processing times, fixation history, tissue characteristics, or otherhistology information can be used to adjust processing to ensure properhistology tissue processing.

At 577, the tissue can be prepared for examination or storage. Thesample can be embedded, sectioned, and transferred onto a microscopeslide for subsequent processing and analyses, such as staining,immunohistochemistry, or in situ hybridization. To section a tissuesample for optical microscope examination, a relatively thin strip oftissue can be cut from a large tissue sample so that light may betransmitted through the thin strip of tissue. A microtome can cut thespecimen into thin sections, for example, slices on the order of about 5microns to about 6 microns thick. Each section can include a portion ofthe tissue sample and some of the embedding material. The microtome andany other equipment (e.g., a staining station, an embedding station, anoven, etc.) used in the processing system 556 can include communicationdevices to read and/or write information to the specimen holder.

The tissue sample can be transferred onto a microscope slide, which caninclude machine-readable code. In some embodiments, the cut sections arefloated on water to spread or flatten the sections. If the sections arepieces of paraffin embedded tissue, the sections can be floated on awarm bath to keep the sections in generally flat configurations, therebyreducing or preventing folding, creasing, or bending. A microscope slideis inserted into the warm bath. A front surface of the slide is used topick up the tissue samples. To examine multiple tissue samples (e.g., aset of tissue samples, each taken at a different location in a subject)using a single slide, a plurality of the tissue samples may besequentially floated onto the slide. These wet slides are then driedusing the slide dryer and coverslipped.

FIG. 25 shows an analyzer 598 that includes a transmitter unit 599 withan array of transmitters 600 a, 600 b, 600 c, 600 d (collectively 600)and a receiver unit 601 with an array of receivers 602 a, 602 b, 602 c,602 d (collectively 602). Transmitters 600 are aligned with respectivereceivers 602. The pairs of transmitters 600 and receivers 602 canmonitor different sections of a specimen 604. The number oftransmitters/receivers, positions of the transmitters/receivers, and thespatial resolution of the analyzer 598 can be selected based on the sizeof the specimen 604. In order to expand the spatial resolution forrelatively small biopsy cores, the focal diameters of the transmitters600 can be relatively small. In certain embodiments, the focal diameterscan be in a range of about 2 millimeters to about 5 millimeters. Otherranges of focal diameters are also possible. Other means of adjustingthe focal properties may include, without limitation, acoustic lenses orapertures in front of the transmitters/receivers. The focal diameters ofthe transmitters 600 can overlap to ensure that the entire specimen 604is analyzed. In other embodiments, the focal diameters of thetransmitters 600 can be spaced apart from one another.

To analyze the specimen 604, a specimen holder 606 can be moved througha gap 608 between the transmitter unit 599 and the receiver unit 601. Insome embodiments, the specimen holder 606 is moved through the gap 608using a transport apparatus. In other embodiments, the specimen holder606 is manually inserted into the gap 608.

FIG. 26 shows an analyzer 620 with a transmitter unit 621 and a receiverunit 623. The transmitter unit 621 includes transmitters 622 a, 622 b,622 c, 622 d (collectively 622). The receiver unit 623 includesreceivers 624 a, 624 b, 624 c, 624 d (collectively 624). The illustratedlinear array of transmitters 622 and linear array of receivers 624 canscan a specimen 630.

Different combinations of transmitters and receivers can be used toprovide different sound paths through tissue samples. As shown, atransmitter 640 can communicate with a receiver 642 such that the soundpaths between the transmitters 622 and receivers 624 is generallyperpendicular to a sound path between the transmitter 640 and thereceiver 642. Thus, measurements can be taken in different directions.Such embodiments are well suited for analyzing specimens withanisotropic properties. The number, types, orientations, and positionsof transmitters/receivers can be selected based on the characteristicsof the specimen.

FIG. 27 shows a specimen holder 700 that includes plates 710, 712 thatare spaced apart and generally parallel to one another. Apertures 702,704 facilitate delivery of acoustic waves to a tissue sample. A specimencan be sandwiched between the plates 710, 712 and held in asubstantially flat configuration. Acoustic energy can travel generallyperpendicular to the plates 710, 712 and can pass through the alignedapertures 702, 704.

Barrier elements 714, 716 can block the apertures 702, 704,respectively. Each of the barrier elements 714, 716 can include, withoutlimitation, a mesh, a perforated material, a web, a grate, a screen,foil, fabric, or any other structure or material through which acousticwaves can travel with minimal, limited, or substantially no attenuation.The barrier elements 714, 716 can thus keep the specimen within thespecimen holder 700. The barrier elements 714, 716 can also be permeableto ensure that a sufficient amount of the specimen is contacted by theprocessing media. In some embodiments commercially available biopsytissue cassettes may be utilized.

FIGS. 28 and 29 show a specimen holder 800 that is generally similar tothe specimen holder 700, except as detailed below. FIG. 29 shows thespecimen holder 800 holding a specimen 810. The specimen holder 800includes transmitters 802 a, 802 b, 802 c, 802 d (collectively 802) andreceivers 804 a, 804 b, 804 c, 804 d (collectively 804). Thetransmitters 802 and receivers 804 can contact or be proximate to thespecimen 810 to minimize signal attenuation and other problems oftenassociated with transmitting across relatively large distances, and tominimize or limit attenuation attributable to processing media (e.g., ifa gap is formed between the specimen 810 and the walls of the holder800).

The transmitters 802 and receivers 804 can be coupled to a main body812. In certain embodiments, the transmitters 802 and receivers 804 arepermanently coupled to or integrated into the main body 812. In otherembodiments, the transmitters 802 and receivers 804 are removablycoupled to the main body 812 to allow components to be interchanged orremoved for inspection, maintenance, or the like. To facilitate physicalcontact between the specimen 810 and processing media, the specimenholder 800 can have any number of apertures and can be made of apermeable or semi-permeable material.

The tissue sample holder 700 of FIG. 27 and the specimen tissue holder800 of FIGS. 28 and 29 can be used with the processing system 100 ofFIGS. 1 and 2, the processing system 450 of FIG. 18, the processingsystems 560 of FIGS. 18-20, the processing system 550 of FIG. 21, etc.Processing systems can thus be configured to receive a wide range ofdifferent types of specimen holders with or without transmitters orreceivers, sensors, apertures, or the like.

FIGS. 30-36 show measurements generated from a processing systemanalyzing specimens. FIGS. 30 and 31 show measurements taken with NBF atroom temperature (e.g., about 20° C.). A heated bath of NBF was used toobtain the measurements of FIGS. 32-35. Heated baths can be used toreduce fixation times. FIG. 36 shows a negative control run in water.

Referring to FIG. 30, beef muscle was cut into approximately 4 mm to 5mm thick pieces and fixed in time increments of about less than 1 hour,2 hours, 4 hours, 6 hours, and 24 hours. TOF was measured while thefixative was kept at room temperature. As shown, equivalent sound speedchange was observed from about 1,580 m/s to about 1,610 m/s.

FIG. 31 shows fixation time versus sound speed and relative TOF change.The measurements were obtained using a sample of beef muscle with athickness of about 4 mm. Inline monitoring was used to monitor fixationin a bath of NBF for about 21 hours. Equivalent sound speed change wasobserved from about 1,520 m/s to about 1,580 m/s.

FIG. 32 shows fixation time versus signal amplitude and TOF change in aheated bath of NBF. A tissue sample of beef muscle tissue was cut acrossits fibers. The tissue sample had a thickness of about 4 mm and wasfixed in a heated bath of NBF. The NBF bath was kept at a temperature ofabout 47° C. with about +/−1° C. control.

FIG. 33 shows fixation time versus signal amplitude and TOF change offat tissue in a heated bath of NBF. The fat tissue had a thickness ofabout 4 mm. The heated NBF bath was maintained at a temperature of about47° C. with about +/−1° C. temperature control.

FIG. 34 shows fixation time versus signal amplitude and TOF change ofliver tissue in a heated bath of NBF. A sample of liver tissue with athickness of about 4 mm was fixed in the heated NBF bath maintained atabout 47° C. with about +/−1° C. temperature control.

FIG. 35 shows fixation time versus signal amplitude and TOF change ofhuman tonsil tissue in a heated bath of NBF. The tissue sample was fixedusing a heated NBF bath maintained at about 47° C. with about +/−1° C.temperature control.

FIG. 36 shows fixation time versus signal amplitude and TOF change ofmuscular beef tissue in a negative control bath of deionized water. Thetissue sample had a thickness of about 4 mm. The bath was heated andmaintained at about 47° C. with about +/−1° C. temperature control.

Two protocols were used to analyze different types of tissue. In oneprotocol, different samples were fixed for different lengths of timesand kept at about room temperature. Signal amplitude was measured inclose succession. In the other protocol, the same samples werecontinuously monitored and kept at elevated temperatures until signallevels reached a plateau.

Both protocols produced similar results, with the elevated temperatureprocessing providing faster fixation. The measurements (e.g., soundspeed measurements) at higher temperatures are subject to morefluctuations due to temperature gradients in the media between thetransmitter and the receiver and warm-up effects from the sample tissueand the specimen holder, which were initially introduced at roomtemperature and had to equilibrate. In another protocol, a specimen andspecimen holder were briefly (e.g., about 5 minutes to about 10 minutes)warmed up externally to about 47° C. before being inserted into themeasurement channel with similar results.

Results of TOF measurements and signal attenuation are shown in FIGS. 37and 38 for comparison between different types of tissue. FIG. 37 showsfixation time versus signal amplitude for beef in water (which serves asa negative control) and fat, beef, liver, and tonsil in a fixative. Thefixative was maintained at a temperature of about 50° C. There is anincrease of the received amplitude in fatty tissue. This may be due tobetter transmission capabilities, changes in density, combinationsthereof, or the like due to the perfusion of the fixative and resultingcross-linking.

FIG. 38 shows fixation time versus change of TOF for different types oftissue maintained at about 50° C. The fatty tissue responded withexponential decay of the sound speed change during perfusion and/orfixation. This may be because of a negative temperature coefficient offat and the warming effects of the tissue due to elevated temperaturetesting. The muscular tissue responded with an exponential growth changeof the sound speed mostly due to cross-linking. The growth change mayalso be increased due to elevated temperatures. FIGS. 30 and 31 show asimilar increase of about 60 ns to about 100 ns (or about 60 m/s insound speed) observed at room temperature, and may be related tofixation.

Table 3 below shows sound speeds in different types of specimens. Thespecimens had a thickness of 4 mm and were fixed with a fixativemaintained at about 47° C.

TABLE 3 Unfixed Speed at Fixed Speed Signal 47° C. at 47° C. Sound speedAmplitude Tissue type [m/s] [m/s] change [m/s] Change [%] Fat (beef)1,687 1,387 −308 +381%  Muscle (beef) 1,618 1,681 +63 −58% Liver (calf)1,767 1,737 −30 −38% Tonsil (human) 1,672 1,702 +30 +27%

If the tissue type of a specimen is known, the sound speed changes(e.g., increases in sound speed, decreases in sound speed, orcombinations thereof) can be used to determine the tissue type. Forexample, if a specimen of an unknown tissue type has an unfixed speed ofabout 1,687 m/s and the sound speed which decreases as the tissue isfixed, it can be concluded that the tissue may be fat tissue from beef.Of course, the unfixed sound speed of the tissue can be compared to thefixed sound speed to determine the tissue type with a high degree ofaccuracy. Different types of tissue samples have differentcharacteristic sound speeds.

Samples can be pre-treated to facilitate fixation (e.g., enhancefixation consistency, reduce fixation time, etc.) and/or monitoring. Insome protocols, a sample can be soaked in media to manage the effects ofperfusion through the sample. If the sample is fixed using formalin, thesample can be pre-soaked in formalin to ensure sufficient diffusion offormalin into the inner sample regions without substantial amounts ofcross-linking. FIG. 39 shows time versus a TOF signal for differenttissue samples. The pre-soaked tissue sample was submersed in coldformalin at 4° C. for about 2 hours. The pre-soaked sample was thensubmerged in high temperature formalin bath (e.g., a bath of 10% NBF atabout 45° C.) to cause cross-linking and accelerate the fixationprocess. The pre-soaked curve shows that the TOF signal graduallydecreases as the sample is fixed. If the sample is processed withglycerol solution, the sample can be pre-soaked in glycerol solution orother type of media with characteristics similar to the characteristicsof glycerol solution.

Pre-soaking can minimize, limit, or substantially eliminate the effectsof water displacement that significantly changes the acousticcharacteristics of the tissue sample. A comparison of the pre-soakedcurve (or media delivery curve) and the not pre-soaked curve shows thatpre-soaking limits or substantially eliminates changes in TOFattributable to media perfusion causing displacement of lower densitywater in the tissue. The initial increase in phase comparison data forthe not pre-soaked tissue may be caused by media perfusion (e.g.,formalin diffusion) into the tissue, thereby displacing lower densitywater with higher density formalin (e.g., due to contained phosphates).The displacement phase is typically followed by the cross-linking phase,as indicated by rapidly declining or increasing TOF signal. A wide rangeof different types of fluid perfusion processes can be monitored becausemost processing media causes a density change in the sample.

Temperature changes in the tissue samples can affect TOF measurements.The samples can be at a temperature that is generally equal to thetemperature of the media to minimize, limit, or substantially eliminatechanges in TOF attributable to density changes caused by temperaturechanges. If the sample is at a different temperature than thetemperature of the media, thermal equilibration can be accounted forbecause thermal equilibrium can be achieve within a few minutes aftersubmersion in the warm solution. For example, if a sample at 4° C. issubmerged in a warm formalin bath (e.g., a bath at 45° C.), the samplecan reach thermal equilibrium in less than about five minutes. Based onthe tissue size and characteristics (e.g., thermal characteristics), thetime to reach thermal equilibrium can be estimated.

Processes that cause changes to tissue structure can be monitored usingthe TOF measurements. For example, a dehydration process can causemeasurable mechanical changes in the tissue. FIG. 40 shows time versusTOF signal for a human tonsil. The dehydration processing causessignificant changes in TOF greater than the changes of TOF caused byfixation. Human tonsil was dehydrated using a 70% ethanol solution byvolume and further dehydrated using a 100% ethanol solution by volume.The TOF signals shown in FIG. 40 were generated using phase detectionalgorithms covering multiple wavelengths.

In other dehydration protocols, tissue is exposed to gradated alcoholconcentrations, to first remove phosphate buffers with a 70%ethanol/water mixture, followed by additional steps in 95% and 100%ethanol by volume to further dehydrate the fixed tissue. The tissue canundergo substantial shrinkage (e.g., more than 10% of its originalvolume). The amount of shrinkage can be determined using TOFmeasurements. The tissue shrinkage can be detected by TOF monitoring dueto the resulting change in the tissue sample (e.g., tissue hardening,tissue shrinkage, etc.).

Monitoring can be used to evaluate whether samples are properlyprocessed. FIG. 41 shows time versus TOF signals of an alcoholdehydration process. The alcohol dehydration can rely on sufficientcross-linking established during the fixation process. The resultingtissue compression during the dehydration processes can be empiricallyknown to produce more disruptive tissue effects (such as tearing andcell and nucleus contraction) when the fixation step is omitted or tooshort. FIG. 41 shows differences in the TOF signal in 70% alcohol byvolume of insufficiently fixed tissue versus properly fixed tissue. Theoverall process time of unfixed tissue in alcohol is significantlylonger and the observed TOF changes are much larger than in fixed tissue(after 24 hours of standard fixation).

Samples can be processed successively in different media to enhance TOFmeasurements. A first dehydration process can be performed in apreconditioning media. For example, a sample can be submerged in a bathof 70% alcohol by volume for about 15 minutes. The partially dehydratedsample is then subjected to a second dehydration process involvingsubmerging the partial dehydrated sample in a bath of 100% alcohol byvolume. As shown in FIG. 42, preconditioning produces a much larger TOFresponse in the 100% alcohol bath as compared to a sample processed in abath of 70% alcohol for about 2 hours. The response in tissuecompression is likely much higher when skipping or performing for toolittle time the 70% alcohol step.

Compensation protocols can be used to minimize, limit, or substantiallyeliminate unwanted noise cause by the environmental factors. Theenvironmental factors may include, without limitation, temperaturechanges due to the ambient temperature, evaporative losses, mediadensity changes (e.g., due to chemical reactions), or the like. If thetemperature of the media fluctuates, the density of the media can alsofluctuate and lead to noise in the TOF measurements. A thermal device(e.g., heating/cooling device) can keep the media within a desiredtemperature range or at a desired temperature. Additionally, a containerholding the media can be thermally insulated to minimize or limittemperature changes.

The containers can be closed to avoid evaporative losses to minimize,limit, or substantially avoid drift. Evaporation of the media can resultin a gradual change in TOF over time. For example, a total change in TOFof about 25 nanoseconds can result from about 15 hours of evaporation.Lids or covers can be placed on the containers to avoid or limitevaporation. Alternatively or additionally, media can be pumped into acontainer to maintain a desired characteristic of the media.

Compensation schemes can be used to minimize, limit, or substantiallyeliminate environmental influences by using a reference channel (i.e., aposition where data is taken, but the tissue or cassette is not in theway of the beam). Data values at this position can be subtracted fromvalues at target positions.

A wide range of signal processing routines can be used to analyze thesignals discussed herein. Filtering routines, compression routines(e.g., true pulse compression routines), cross-correlation routines,auto correlation signal recovery (especially in noisy environments), orthe like can be utilized. Signal processing is especially well suitedwhen samples are in relatively small containers in which there may bestanding waves, reflections, and echoing. Signal processing routines canthus be selected to significantly improve signal-to-noise ratios.

FIG. 39 shows the time of flight signal in a reference sample, apre-soak sample, and a sample that has not been subjected to a pre-soakprocess. As shown with the no pre-soak curve, the TOF signal generallyincreases immediately after submerging a tissue sample into anon-buffered formula and bath at a temperature of about 45° C. Afterabout six to ten minutes, the TOF signal gradually decreases similar tothe pre-soaked specimen. As indicated in FIG. 9, the TOF signals betweenthe pre-soaked and non-pre-soaked samples can generally correspond towater being displaced by the formalin. Because the formalin and thewater have different sound transmissibilities, the TOF signals change asthe formalin replaces the interstitial in the sample.

FIG. 43 shows TOF of different liquids at the same temperature. Becausespeed of sound through a liquid is dependent on the liquid's bulkmodulus. TOF measurements can be used to determine the composition ofliquids at a known temperature. Additionally, liquid's TOF can bemeasured to predict diffusion rates through a solid tissue sample. Asshown in FIG. 43, the TOF for 10% NBF can be about 2.1% to about 4.4%greater than the TOF signal of 20% NBF. The TOF of water can be greaterthan about 3.2% to about 8% than the TOF of 10% NBF. Based on a knowndistance of travel by the acoustic waves, the type of fluid can bedetermined based on, for example, a TOF changes, temperaturemeasurements, or the like. A diffusion algorithm can use a fluid's bulkmodulus, acoustic characteristics, or other properties to determine thediffusion state in the tissue sample.

FIG. 44 is a plot of time versus a change in TOF for tissue samples(cores of human tonsil with approximately 6 mm diameters) immersed inice water (about 1° C. to about 4° C.) and different fixatives. The TOFcan decrease when tissue is placed in the ice water, from about zeronanoseconds to about −13 nanoseconds for a period of about 2.8 hours. Asshown in FIG. 44, the TOF can decrease generally linearly with respectto time. The TOF can decrease non-linearly from about −44 nanoseconds toabout −52.5 nanoseconds when the sample is placed in 10% NBF at 40° C.for about 2.8 hours. A desired level of diffusion (or perfusion) cancorrespond to a target rate of change of the TOF. For example, when theTOF rate of change reaches a target TOF rate of change, the diffusionprocess can be inhibited or stopped by removing the sample from the 10%NBF solution. Even though the specimen is removed from the 10% NBFsolution, a small amount of 10% NBF may continue to diffuse through thetissue. For 20% NBF, the TOF can decrease to about −66 nanoseconds toabout −82 nanoseconds for a processing period of about 2.8 hours.

Processing systems and analyzers disclosed herein can be used to monitormovement of fluids through solid tissue samples in real-time. Forexample, the processing system 100 discussed in connection with FIGS. 1and 2 can be used to monitor diffusion of media 170 through the specimen150. If the media 170 is a fixative, it can be at a relatively lowtemperature to inhibit or minimize cross-linking such that changes inTOF signals primarily indicate media 170 displacing interstitial fluidrather than changes in TOF associated with cross-linking.

The computing device 160 of FIG. 1 can contain instructions for TOFacquisition schemes that determine center frequencies for TOFmeasurements. The scheme can include performing an initial centerfrequency scan to analyze phase relationships between transmitted andreceived ultrasound wave packets. This analyzing process can beperformed to determine an optimal center frequency at which the phasedifference observed by the system 100 is in a general midpoint of itscomparison range. The new center frequency scheme can be a baseline andutilized as a single frequency scheme. This allows for rapid TOFacquisitions by producing an average of 100 to 1,000 phase comparisonsat a rate of about 60 to 80 times per second. In some methods, thecomparisons can be formed at a rate of about 70 times per second forreal-time monitoring. Accordingly, the system 100 can allow monitoringof target changes up to about 120 nanoseconds at 4 MHz based on a rangeof phase comparator circuitry. Additionally, the center frequency can beadjusted to compensate for significant changes in TOF attributable to,for example, temperature changes, such as ambient temperature changes.The center frequency can be used to select a new midrange setting, ifneeded or desired. Based on the selected center frequency, the TOF canbe monitored. The computing device 160 can include other TOF samplingschemes.

Referring to again to FIG. 19, the system 560 of can monitor diffusionbased on TOF. The container 592 a can hold fixative at a temperatureless than about 15° C. In one procedure, the fixative can be within atemperature range of about −20° C. to at least 15° C., preferablygreater than 0° C. to an upper temperature more typically about 10° C.,and even more typically from about 3° C. to 5° C. to preventcross-linking to a significant extent. The tissue sample can remain incontact where the fixative composition for about 15 minutes to about 4hours. In some procedures, the contact time is about 15 minutes to 3hours. Other times can also be used, if needed or desired. In someembodiments, the tissue samples with thicknesses of about 4 mm can beprocessed at fixative temperatures at about room temperature in the samecontainer, and monitored for diffusion and cross-linking for a period ofabout 4 hours to about 24 hours. Thicker tissue samples (e.g., samplesfrom resected materials) may need additional time for diffusion andcross-linking. For example, such tissue samples may be diffused up tomany days using a room temperature fixative. These diffusion processescan also be monitored.

After the desired diffusion level is achieved, the transport apparatus586 can move the tissue samples to the container 592 b. The container592 b can contain a warm fixative. In some embodiments, the fixative inthe container 592 a is substantially the same as the fixative incontainer 592 b. In other embodiments, concentrations and/ortemperatures of fixatives in the container 592 a, 592 b can bedifferent. The temperature of the fixative in container 592 b can be ina range of about 22° C. to about 50° C. After a desired level offixation is achieved, tissue samples can be moved from container 592 b.

FIG. 45 is a plot of time versus a change in TOF for tissue samplespre-soaking in 10% NBF, 20% NBF, 30% NBF, and 40% NBF. The higherconcentration fixatives (e.g., 40% NBF) provide large TOF changes thatcan be accurately monitored to determine the status of tissue samples.Based on the composition of the fixative and measured TOF, the diffusiontime can be approximated to ensure that the diffusion process iscompleted.

It should be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to an analyzer including “a transmitter” includes a singletransmitter, or two or more transmitters. It should also be noted thatthe term “or” is generally employed in its sense including “and/or”unless the content clearly dictates otherwise.

The various embodiments and features described above can be combined toprovide further embodiments. These and other changes can be made to theembodiments in light of the above-detailed description. In general, inthe following claims, the terms used should not be construed to limitthe claims to the specific embodiments disclosed in the specificationand the claims, but should be construed to include all possibleembodiments along with the full scope of equivalents to which suchclaims are entitled. Accordingly, the claims are not limited by thedisclosure.

We claim:
 1. A method for preparing a tissue sample having a thicknessof not more than 5 mm, comprising: immersing a tissue sample, which isin an unfixed state, in a fixative having a temperature ranging frombetween about −15° C. to about 15° C.; monitoring diffusion of thefixative across a thickness of the tissue sample by continuouslymonitoring a rate of change in time of flight of acoustic waves thattravel through the tissue sample, wherein the rate of change in time offlight (TOF) is calculated by a system comprising a computing devicecommunicatively coupled to a transmitter and a receiver, wherein: saidtransmitter outputs acoustic waves that pass through the tissue sample,said receiver receives the acoustic waves that have passed through thetissue sample and sends signals to the computing device in response tothe received energy, and the computing device analyzes signals from thereceiver to calculate the TOF; continuing diffusion at least until atarget rate of change of TOF is achieved; and increasing the temperatureof the fixative in which the tissue sample is immersed to a temperaturein a range from about 22° C. to about 50° C. after the fixative hasdiffused across most of a thickness of a tissue sample.
 2. The method ofclaim 1, wherein the fixative comprises liquid formalin.
 3. The methodof claim 1, wherein monitoring the diffusion of the fixative includesdetecting displacement of interstitial fluid in the tissue sample byacoustically evaluating the tissue sample while the fixative movesthrough the tissue sample.
 4. The method of claim 1, wherein monitoringthe diffusion of the fixative comprises simultaneously measuringdiffusion of the fixative and cross-linking performed at the sametemperature.
 5. The method of claim 1, wherein monitoring the diffusionincludes measuring time of flight of the acoustic waves at least 50times per second.
 6. The method of claim 1, wherein time of flight (TOF)is determined according to Formula I:TOF=t1+t2+t3  (I) wherein: t1 is an individual travel time of theacoustic waves traveling from the transmitter to the tissue sample, t2is the individual travel time of the acoustic waves traveling throughthe tissue sample, and t3 is an individual travel time of the acousticwaves traveling from tissue sample to receiver.
 7. The method of claim1, further comprising measurement of a reference TOF and compensation ofthe TOF measurements of the tissue sample based on the reference TOF. 8.The method of claim 1, wherein the system allows monitoring of targetchanges up to about 120 nanoseconds at 4 MHz.
 9. The method of claim 1,wherein the tissue sample is immersed in a cold fixative at atemperature of from 0 ° C. to 10 ° C.
 10. A method for preparing a fixedtissue sample having a thickness of not more than 5 mm, comprising:immersing a tissue sample, which is in an unfixed state, in a coldfixative wherein the cold fixative is at a temperature ranging frombetween about 0° C. to about 10° C.; monitoring a rate of change in timeof flight of acoustic waves that travel through the tissue sample,wherein the rate of change in time of flight (TOF) is calculated by asystem comprising a computing device communicatively coupled to atransmitter and a receiver, wherein the transmitter outputs acousticwaves that pass through the tissue sample, the receiver receives theacoustic waves that have passed through the tissue sample and sendssignals to the computing device in response to the received energy, andthe computing device analyzes signals from the receiver to calculate theTOF; continuing diffusion of the cold fixative at least until a targetrate of change of TOF is achieved; and after the target rate of changeof TOF is achieved, heating the tissue sample to promote cross-linkingof the tissue sample at a temperature ranging from between about 22° C.to about 50° C., wherein the target rate of change is a rate of changeindicative that the cold fixative has diffused across most of athickness of the tissue sample.
 11. The method of claim 10, furthercomprising embedding said tissue sample in paraffin after completion ofthe fixation process.
 12. The method of claim 11, further comprisingsectioning said embedded tissue.
 13. The method of claim 1, wherein thecomputing device is configured in a transmission mode.
 14. The method ofclaim 13, wherein the computing device is configured in a shadowedtransmission mode.
 15. The method of claim 13, wherein the computingsystem calculates a reference TOF measurement of the fixative andcompensates the TOF measured through the sample based on the referenceTOF measurement.
 16. The method of claim 10, wherein the fixativecomprises liquid formalin.
 17. The method of claim 10, whereinmonitoring the diffusion of the fixative includes detecting displacementof interstitial fluid in the tissue sample by acoustically evaluatingthe tissue sample while the fixative moves through the tissue sample.18. The method of claim 10, wherein monitoring the diffusion of thefixative comprises simultaneously measuring diffusion of the fixativeand cross-linking performed at the same temperature.